KR101869268B1 - Cell Permeable Fusion Protein for Strengthening Regenerative Potential of Stem Cells - Google Patents

Cell Permeable Fusion Protein for Strengthening Regenerative Potential of Stem Cells Download PDF

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KR101869268B1
KR101869268B1 KR1020110051621A KR20110051621A KR101869268B1 KR 101869268 B1 KR101869268 B1 KR 101869268B1 KR 1020110051621 A KR1020110051621 A KR 1020110051621A KR 20110051621 A KR20110051621 A KR 20110051621A KR 101869268 B1 KR101869268 B1 KR 101869268B1
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정종평
박윤정
이주연
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서울대학교산학협력단
주식회사 나이벡
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Abstract

본 발명은 줄기세포 재생 강화용 세포 침투성 융합 단백질에 관한 것으로, 더욱 자세하게는 줄기세포의 분화촉진능, 세포 사멸 억제능, 줄기세포의 기능성 유지 및 스트레스에 의해 억제된 줄기세포의 기능성을 회복하기 위한 줄기세포 재생 강화용 세포 침투성 융합 단백질에 관한 것이다.The present invention relates to a cell-penetrating fusion protein for enhancing stem cell regeneration, and more particularly, to a stem cell regeneration ability, a cell death-suppressing ability, a stem for restoring the function of a stem cell, And a cell permeable fusion protein for enhancing cell regeneration.

Description

줄기세포 재생 또는 증식능 향상용 세포 침투성 융합 단백질 {Cell Permeable Fusion Protein for Strengthening Regenerative Potential of Stem Cells}[0001] Cell Permeable Fusion Proteins for Strengthening Regenerative Potential of Stem Cells [

본 발명은 줄기세포 재생 또는 증식능 향상용 세포 침투성 융합 단백질에 관한 것으로, 더욱 자세하게는 줄기세포의 분화촉진능, 세포 사멸 억제능, 줄기세포의 기능성 유지 및 스트레스에 의해 억제된 줄기세포의 기능성을 회복하기 위한 줄기세포 재생 또는 증식능 향상용 세포 침투성 융합 단백질에 관한 것이다.
The present invention relates to a cell-permeable fusion protein for enhancing stem cell regeneration or proliferative activity, and more particularly, to a cell-permeable fusion protein capable of regenerating the function of stem cells inhibited by stem cell differentiation promoting ability, cell killing inhibitory ability, The present invention relates to a cell-permeable fusion protein for enhancing stem cell regeneration or proliferation.

중간엽 줄기세포(Mesenchymal Stem Cell)는 배아기에 골격계의 생성 및 성장에 관여하고 성장이 끝나면 골세포 외에 연골세포, 근육세포, 섬유세포 등 여러가지 종류의 골격계 세포들로 분화할 수 있는 능력을 가진 세포를 말하며, 골수기질간세포(Bone Marrow Stromal Stem Cell)라고도 한다. 중간엽세포는 배아줄기세포와는 달리 종양을 유발하거나 또는 윤리적인 제한점이 없어서 임상적 활용성이 높다. The Mesenchymal Stem Cell is involved in the production and growth of the skeletal system in the embryo. When the growth is completed, the mesenchymal stem cell (Mesenchymal Stem Cell) is a cell capable of differentiating into various kinds of skeletal cells such as chondrocytes, muscle cells, And is also referred to as a bone marrow stromal stem cell (Bone Marrow Stromal Stem Cell). Unlike embryonic stem cells, mesenchymal cells have high clinical utility because they induce tumors or have no ethical limitations.

그러나 중간엽 줄기세포의 이식에서 해결해야할 과제는 이식후의 체내에서의 생착 (적응력향상) 및 증식을 원활하게 하는 기술의 개발, 그리고 원하는 조직으로의 선택적인 분화기능성 확보이다. 이식후 체내에서의 적응력 향상을 위해서는 이식후 면역관련 약제를 투여하기는 하지만 이는 이식받는 주체의 적응력을 위한 것이지 이식세포의 적응력 향상을 위한 물질은 보고된 바 없다. However, the problem to be solved in the transplantation of mesenchymal stem cells is to develop a technique for facilitating engraftment (enhancement of adaptation) and proliferation in the body after transplantation, and securing selective differentiation function to a desired tissue. In order to improve the adaptive capacity in the body after transplantation, post-transplant immunotherapy is administered, but this is for the adaptability of the subject to be transplanted, and no substance has been reported to improve the adaptability of the transplantable cell.

이식시의 주변 조직환경에 의해 이식된 줄기세포는 Senescence를 겪게 되면서 사멸하게 되면서 이식의 효과를 기할 수 없게 된다. 또한 이식된 줄기세포의 원활한 증식 역시 재생을 위해서 선결조건이라고 할 수 있다. 생착과 증식에 더 나아가 원하는 목적조직, 본 발명에서는 골조직으로의 선택적인 분화를 촉진하는 물질 역시 보고된 사례는 없다. 중간엽 줄기세포에서 조골세포와 지방세포로 분화되는 결정적 전사인자는 Runx2와 표백작용 성장 활성화 수용체 PPAR gamma이다. 이들은 중간엽 줄기세포들로 하여금 뼈를 만드는 조골세포 또는 지방세포로 차별화 하도록 유도한다. Runx2는 자체적으로도 외부에서 주입시 뼈로의 이행이 관찰된 단백질이며, 이 외에 여러 전사단백질들이 세포의 골조직으로의 분화를 촉진시키는 것으로 알려져있다. 특히, Hong 등은 14-3-3 이라 불리는 결속 단백질인, PDZ-결속을 유도하는 전사 활동자인 TAZ라는 유전자가 PPAR gamma 전사를 억제하고 Runx2를 활성화한다는 사실을 규명했다 (Hong J.H. et al., Science, 309:1074, 2005). 이러한 단백질은 세포질 내에서 골조직 분화 유전자를 활성화시키는 작용을 한다. 그러나 이들이 특정 목적으로 세포질내로 혹은 조직내로 도입이 되기에는 분자량이 크기 때문에 (분자량이 600 이상인 물질은 세포막을 통과하기가 거의 불가능하다), 세포내 도입수송체의 활용이 절실한 상황이다. The stem cells transplanted by the surrounding tissue environment at the time of transplantation die as they undergo senescence, and can not achieve the transplantation effect. In addition, smooth proliferation of transplanted stem cells is also a prerequisite for regeneration. There are no cases reported in the present invention for promoting selective differentiation into bone tissue, further aiming at engraftment and proliferation. The crucial transcription factor that differentiates into osteoblasts and adipocytes in mesenchymal stem cells is Runx2 and the PPAR gamma binding activator. They induce mesenchymal stem cells to differentiate into osteoblasts or adipocytes that make bone. Runx2 itself is a protein that has been observed to migrate from the outside to the bone at the time of injection, and various transcriptional proteins are known to promote cell differentiation into bone tissue. In particular, Hong et al. Found that a gene called TAZ, a transcriptional activator that induces PDZ-binding, a binding protein called 14-3-3, inhibits PPAR gamma transcription and activates Runx2 (Hong JH et al. Science, 309: 1074, 2005). These proteins act to activate bone differentiation genes in the cytoplasm. However, since they have a high molecular weight to be introduced into the cytoplasm or into tissues for specific purposes (a substance having a molecular weight of 600 or more is almost impossible to pass through the cell membrane), the use of intracellular transporters is urgent.

최근 국내외에서 골다공증, 골절 및 수술 등으로 인하여 손실된 뼈를 회복하기 위한 골형성 촉진제의 개발이 요구되고 있다. 그러나, 현재까지 개발된 비스포스포네이츠(bisphosphonates), 칼시토닌(calcitonin), 에스트라디올(estradiol) 또는 비타민 D와 같은 골형성 촉진제는 골흡수의 억제에 주목적이 있고 이미 상실된 뼈의 재생에는 큰 효과가 없는 실정이다. 이에 골형성을 촉진할 수 있는 새로운 약제를 찾아내기 위하여 많은 노력을 기울이고 있다. Recently, there has been a demand for development of an osteogenesis promoting agent for restoring lost bone due to osteoporosis, fracture and surgery at home and abroad. However, osteogenesis promoters such as bisphosphonates, calcitonin, estradiol or vitamin D, which have been developed so far, are mainly used for inhibiting bone resorption and have a great effect on the regeneration of bone that has already been lost It is absent. Therefore, a lot of efforts are being made to find a new drug that can promote bone formation.

최근 인간 면역결핍 바이러스(Human Immunodeficiency Virus type-1) 단백질의 일종인 Tat(Transactivator of transcription) 단백질은 효율적으로 세포막을 통과하여 세포질 내로 쉽게 이동한다는 것이 밝혀졌다. 이러한 기능은 Tat 단백질의 중간부위인 단백질 형질도입 부위(PTD, Protein Transduction Domain)의 특성 때문에 나타나며 아직 그 정확한 메카니즘은 알려지지 않은 상태이다 (Frankel, A.D. and Pabo, C.O., Cell, 55:1189, 1988; Green, M. and Loewenstein, P.M., Cell, 55:1179, 1988; Ma, M. and Nath, A., J. Virol., 71:2495, 1997; Vives, E. et al., B. J. Biol. Chem., 272:16010, 1997). Recently, Tat (Transactivator of transcription) protein, a type of Human Immunodeficiency Virus type-1 protein, has been found to be efficiently transferred through the cell membrane and into the cytoplasm. This function is due to the characteristics of the protein transduction domain (PTD), which is the middle part of the Tat protein, and its precise mechanism is unknown yet (Frankel, AD and Pabo, CO, Cell, 55: 1189, 1988; 1997, Vives, E. et al., BJ Biol. Chem. ≪ RTI ID = 0.0 > , 272: 16010,1997).

한편, 종래 PTD를 다른 펩타이드 또는 단백질과 연결시킨 경우, 이러한 융합 단백질의 세포 내 수송이 효율적이라는 것이 밝혀진 이후, PTD를 이용한 다양한 응용이 시도되었으나(대한민국 특허등록 제10-0568457호), 아직까지 중간엽 줄기세포의 생착을 촉진하고 증식을 유도하는 목적으로 전사인자인 NF-Ya, 또는 항산화기능성 단백질 (예: SOD, Thioredoxin)을 세포투과성 펩타이드와 결합시켜 조직공학에 적용하려는 시도는 없었다.On the other hand, it has been found that when PTD is linked to another peptide or protein, intracellular transport of such a fusion protein is efficient, various applications using PTD have been tried (Korean Patent Registration No. 10-0568457) There has been no attempt to apply NF-Ya, a transcription factor, or an antioxidant functional protein (for example, SOD, Thioredoxin) to a cell permeable peptide for tissue engineering, in order to promote the engraftment of the stem cells and induce proliferation.

이에 본 발명자들은 줄기세포 재생 또는 증식능 향상용 세포 침투성 단백질을 개발하기 위하여 예의 노력한 결과, 항산화 단백질 또는 증식 향상능을 가지는 단백질의 아미노 말단에 세포 침투성 펩타이드를 결합시킨 융합 단백질을 제조하고, 상기 융합 단백질이 줄기세포의 이식시 이식 성공률을 높이고, 골조직재생을 선택적으로 유도하여 골질환 예방 및 치료에 사용될 수 있으며, 지방세포를 억제하는 단백질을 세포 내로 효율적으로 운반할 수 있고, 합성이 용이하고, 독성에 대한 염려가 전혀 없다는 것을 확인하고, 본 발명을 완성하게 되었다.
Accordingly, the present inventors have made intensive efforts to develop a cell permeable protein for improving stem cell regeneration or proliferative capacity. As a result, they have found that a fusion protein comprising a cell permeable peptide bound to the amino terminal of an antioxidant protein or a protein having a proliferation- It can be used for prevention and treatment of osteopathy by selectively inducing regeneration of bone tissue by transplantation of stem cells, and it is possible to efficiently transport proteins inhibiting adipocytes into cells, easy to synthesize, toxic And that the present invention has been completed.

본 발명의 목적은 줄기세포 재생 또는 증식능 향상용 세포 침투성 융합 단백질을 제공하는 데 있다.
It is an object of the present invention to provide a cell-penetrating fusion protein for stem cell regeneration or proliferative capacity enhancement.

상기 목적을 달성하기 위하여, 본 발명은 항산화 단백질 또는 증식 향상능을 가지는 단백질의 아미노 말단에 세포 침투성 펩타이드가 결합되어 있는 줄기세포 재생 또는 증식능 향상용 세포 침투성 융합 단백질을 제공한다.In order to achieve the above object, the present invention provides a cell permeable fusion protein for enhancing stem cell regeneration or proliferative activity, wherein a cell permeable peptide is bound to an amino terminal of an antioxidant protein or a protein having proliferation improving ability.

본 발명은 또한, 항산화 단백질과 증식 향상능을 가지는 단백질과의 융합 단백질의 아미노 말단에 세포 침투성 펩타이드가 결합되어 있는 줄기세포 재생 또는 증식능 향상용 세포 침투성 융합 단백질을 제공한다.The present invention also provides a cell-permeable fusion protein for enhancing stem cell regeneration or proliferation, wherein a cell permeability peptide is bound to the amino terminal of a fusion protein of an antioxidant protein and a protein having proliferation-improving ability.

본 발명은 또한, 세포 침투성 펩타이드 형질 도입 부위를 coding 하는 cDNA; 항산화 단백질 형질 도입 부위를 coding 하는 cDNA 및/또는 증식 향상능을 가지는 단백질 형질 도입 부위를 coding 하는 cDNA; 골조직 분화기능성 전사인자 단백질을 coding 하는 cDNA; 및 6개의 히스티딘에 대한 cDNA를 포함하고 있으며, 줄기세포 재생 또는 증식능 향상용 융합 단백질의 발현 벡터를 제공한다.The present invention also relates to a cDNA encoding a cell penetrating peptide transduction site; A cDNA coding for an antioxidant protein transduction site and / or a cDNA coding for a protein transduction site having a proliferation improving ability; CDNA coding for a bone-differentiation functional transcription factor protein; And six histidine cDNAs, and provides an expression vector of a fusion protein for enhancing stem cell regeneration or proliferation.

본 발명은 또한, 세포 침투성 펩타이드 형질 도입 부위를 coding 하는 cDNA ; 항산화 단백질 형질 도입 부위를 coding 하는 cDNA 및/또는 증식 향상능을 가지는 단백질 형질 도입 부위를 coding 하는 cDNA; 골조직 분화기능성 전사인자 단백질을 coding 하는 cDNA; 및 6개의 히스티딘에 대한 cDNA를 포함하고 있으며, 줄기세포 재생 또는 증식능 향상을 보이는 융합 단백질의 발현 벡터로 형질전환된 재조합 미생물을 제공한다.The present invention also relates to a cDNA encoding a cell penetrating peptide transduction site; A cDNA coding for an antioxidant protein transduction site and / or a cDNA coding for a protein transduction site having a proliferation improving ability; CDNA coding for a bone-differentiation functional transcription factor protein; And cDNA for six histidines, and provides a recombinant microorganism transformed with an expression vector of a fusion protein showing an improvement in stem cell regeneration or proliferation.

본 발명은 또한 세포 침투성 펩타이드 형질 도입 부위를 coding 하는 cDNA ; 항산화 단백질 형질 도입 부위를 coding 하는 cDNA 및/또는 증식 향상능을 가지는 단백질 형질 도입 부위를 coding 하는 cDNA; 골조직 분화기능성 전사인자 단백질을 coding 하는 cDNA; 및 6개의 히스티딘에 대한 cDNA를 포함하고 있으며, 줄기세포 재생 또는 증식능 향상을 보이는 융합 단백질의 발현 벡터로 형질전환된 재조합 미생물을 배양하여 줄기세포 재생 또는 증식능 향상용을 보이는 융합 단백질을 발현시키는 단계; 및 상기 발현된 융합 회수하는 단계를 포함하는 줄기세포 재생 또는 증식능 향상용 융합 단백질의 제조방법을 제공한다.The present invention also relates to a cDNA encoding a cell penetrating peptide transduction site; A cDNA coding for an antioxidant protein transduction site and / or a cDNA coding for a protein transduction site having a proliferation improving ability; CDNA coding for a bone-differentiation functional transcription factor protein; And expressing a fusion protein which is capable of regenerating stem cell or proliferating ability by culturing a recombinant microorganism transformed with an expression vector of a fusion protein comprising the cDNA for 6 histidine and exhibiting an improvement in stem cell regeneration or proliferation ability; And recovering the expressed fusion. The present invention also provides a method for producing a fusion protein for enhancing stem cell regeneration or proliferation.

본 발명은 또한, 세포 침투성 펩타이드 형질 도입 부위를 coding 하는 cDNA ; 항산화 단백질 형질 도입 부위를 coding 하는 cDNA 및/또는 증식 향상능을 가지는 단백질 형질 도입 부위를 coding 하는 cDNA; 골조직 분화기능성 전사인자 단백질을 coding 하는 cDNA; 및 6개의 히스티딘에 대한 cDNA를 포함하고 있으며, 줄기세포 재생 또는 증식능 향상용 융합 단백질의 발현 벡터로 형질전환된 재조합 미생물을 배양하여 줄기세포 재생 또는 증식능 향상용 융합 단백질을 발현시키는 단계; 및 상기 발현된 융합 회수하는 단계를 포함하는 줄기세포 재생 또는 증식능 향상용 융합 단백질의 제조방법에 의해 제조된 융합 단백질을 줄기세포에 도입하는 것을 특징으로 하는 줄기세포의 재생방법을 제공한다.The present invention also relates to a cDNA encoding a cell penetrating peptide transduction site; A cDNA coding for an antioxidant protein transduction site and / or a cDNA coding for a protein transduction site having a proliferation improving ability; CDNA coding for a bone-differentiation functional transcription factor protein; And expressing a fusion protein for enhancing stem cell regeneration or proliferative capacity by culturing a recombinant microorganism transformed with an expression vector of a fusion protein for enhancing stem cell regeneration or proliferative activity comprising cDNA for six histidines; And a step of introducing the fusion protein produced by the method for producing a fusion protein for stem cell regeneration or proliferation enhancement into a stem cell comprising the step of expressing the fusion and recovering the fusion.

본 발명은 또한, 항산화 단백질 또는 증식 향상능을 가지는 단백질과 골조직 분화기능성 전사인자 단백질과의 융합 단백질의 말단에 세포 침투성 펩타이드가 결합되어 있는 골형성 촉진용 세포 침투성 융합 단백질을 제공한다.The present invention also provides a cell permeable fusion protein for promoting bone formation, wherein a cell permeable peptide is bound to the end of a fusion protein of an antioxidant protein or a protein having proliferation-improving ability and a bone-differentiation-functioning transcription factor protein.

본 발명은 또한, 항산화 단백질 또는 증식 향상능을 가지는 단백질과 골조직 분화기능성 전사인자 단백질과의 융합 단백질의 말단에 세포 침투성 펩타이드가 결합되어 있는 골형성 촉진용 세포 침투성 융합 단백질을 유효성분으로 포함하는 골질환 치료용 약학적 조성물을 제공한다.The present invention also relates to a bone marrow-derived bone marrow-derived bone marrow-derived bone marrow-derived bone marrow-derived bone marrow-derived bone marrow-derived bone marrow- A pharmaceutical composition for treating diseases is provided.

본 발명은 또한, 항산화 단백질 또는 증식 향상능을 가지는 단백질과 골조직 분화기능성 전사인자 단백질과의 융합 단백질의 말단에 세포 침투성 펩타이드가 결합되어 있는 골형성 촉진용 세포 침투성 융합 단백질을 유효성분으로 포함하는 골질환 치료용 건강기능성 식품을 제공한다.
The present invention also relates to a bone marrow-derived bone marrow-derived bone marrow-derived bone marrow-derived bone marrow-derived bone marrow-derived bone marrow-derived bone marrow- A health functional food for treating diseases is provided.

본 발명에 따르면, 줄기세포 재생 강화용 세포 침투성 융합 단백질로 줄기세포를 처리할 경우, 줄기세포 내로 쉽게 침투하여 줄기세포의 분화촉진능, 세포 사멸 억제능, 줄기세포의 기능성 유지 및 스트레스에 의해 억제된 줄기세포 기능성의 회복을 촉진하므로 줄기세포의 재생에 유용하다.
According to the present invention, when a stem cell is treated with a cell permeable fusion protein for enhancing stem cell regeneration, it can be easily penetrated into a stem cell and inhibited by stem cell differentiation promoting ability, cell death suppressing ability, stem cell function maintenance and stress It is useful for the regeneration of stem cells because it promotes recovery of stem cell function.

도 1은 LMWP-Thioredoxin-1 복합체의 세포내 투과 능력을 확인한 결과이다.
도 2는 LMWP-Thioredoxin-1 복합체의 생체내 기능성을 규명한 결과이다.
도 3은 LMWP-SOD 융합단백질의 노화 억제 능력을 확인한 결과이다.
도 3A는 LMWP-SOD 융합단백질 처리에 의한 세포의 모양변화이며, 도 3B 및 도 3C는 senesence associated beta-galactosidase 염색한 결과이다.
도 4는 LMWP-SOD 융합단백질의 노화 억제 능력을 확인한 결과이다.
도 4A 및 도 4B는 p53 유전자, p21 유전자의 RT-PCR 결과이며, 도 4C 및 도 4D는 p53 단백질, p21 단백질의 Western blotting 결과이다.
도 5는 LMWP-SOD 융합단백질에 의한 줄기세포 기능성 회복 확인한 결과이다.
도 5A 및 도5B는 석회화능의 변화를 보여주고, 도 5C 및 5D는 경조직 마커 (ALP, type I collagen, osteopontin)의 RT-PCR 결과이다.FIG. 1 shows the results of confirming the intracellular permeability of the LMWP-Thioredoxin-1 complex.
Figure 2 shows the in vivo functionalities of the LMWP-Thioredoxin-1 complex.
FIG. 3 shows the results of confirming the ability of the LMWP-SOD fusion protein to inhibit aging.
FIG. 3A shows changes in cell morphology by treatment with LMWP-SOD fusion protein, and FIGS. 3B and 3C are results of senesence associated beta-galactosidase staining.
Fig. 4 shows the results of confirming the aging-inhibiting ability of the LMWP-SOD fusion protein.
FIGS. 4A and 4B are RT-PCR results of p53 gene and p21 gene, and FIGS. 4C and 4D are Western blotting results of p53 protein and p21 protein.
FIG. 5 shows the results of recovery of stem cell function by the LMWP-SOD fusion protein.
Figures 5A and 5B show changes in calcification ability, and Figures 5C and 5D show RT-PCR results of hard tissue markers (ALP, type I collagen, osteopontin).

다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

본 발명에서 사용되는 "항산화 단백질"은 다양한 원인에 의해 발생하는 활성 산소를 억제하거나 산화적 스트레스로 인한 단백질들의 산화 상태를 환원 상태로 전환시켜서 단백질의 변성 및 기능 상실을 억제하는 단백질을 의미하며, SOD(Superoxide dismutase), Thioredoxin, catalase, glutathion peroxidase, heam-containing peroxidase 및 이의 유사체들을 포함한다.As used herein, the term "antioxidant protein" refers to a protein that inhibits active oxygen generated by various causes or converts oxidation states of proteins due to oxidative stress to a reduced state to inhibit protein denaturation and loss of function. Superoxide dismutase (SOD), thioredoxin, catalase, glutathione peroxidase, heam-containing peroxidase and analogues thereof.

"SOD(Superoxide dismutase, 서열번호 1)"는 자유 라디칼 음이온이 세포에 해로운 영향을 미치는 바, SOD는 자유 라디탈 음이온을 포함하는 초과산화이온을 산소와 과산화수소로 바꿔 줌으로써 독성으로부터 세포를 방어하는 역할을 한다. 산소에 노출되는 거의 모든 세포에서 이러한 SOD에 의한 항산화방어기작이 중요하며, 일부 유산균들의 경우 다른 방어기작을 사용하는 것으로 알려져 있다."SOD (Superoxide dismutase, SEQ ID NO: 1)" has a detrimental effect on free radical anions, and SOD protects cells from toxicity by replacing excess oxidized ions including free radical anions with oxygen and hydrogen peroxide . Antioxidant defense mechanisms by SOD are important in almost all cells exposed to oxygen, and some lactic acid bacteria are known to use other defense mechanisms.

"Thioredoxin(서열번호 2)"는 분자량이 10,000~13,000의 저분자단백을 뜻하며, 리보뉴클레오티드 환원효소가 리보뉴클레오티드를 환원할 때의 프로톤공급체로서 대장균으로부터 분리되었다. 활성중심에 존재하는 1쌍의 시스테인잔기는 원핵생물에서 진핵생물에 이르기까지 보존되어 있고, NADPH와 티오레독신환원효소의 존재하에 표적단백질의 황화물결합을 환원 개열시키는 활성이 있다. 사람 TRX/ADF(성인 T세포백혈병 유래인자)은 세포증식이나 전사인자 제어에도 관여한다."Thioredoxin (SEQ ID NO: 2)" refers to a low molecular weight protein having a molecular weight of 10,000 to 13,000, and ribonucleotide reductase was isolated from E. coli as a proton donor when ribonucleotides were reduced. A pair of cysteine residues in the active center is conserved from prokaryotes to eukaryotes and has the activity of reducing and cleaving sulfide bonds of target proteins in the presence of NADPH and thioredoxin reductase. Human TRX / ADF (adult T cell leukemia-derived factor) is also involved in cell proliferation and transcription factor control.

본 발명에서 사용되는 "증식 향상능을 가지는 단백질"은 줄기세포를 이식한 후, 종래 세포와의 생착을 통하여 적응력을 향상시키고 증식을 원활하게 하는 단백질을 의미하며, HOX4 단백질 및 NF-Ya단백질 등이 있다.As used herein, the term "protein having proliferation-enhancing ability" means a protein that transplants stem cells and then improves adaptability and facilitates proliferation through engraftment with conventional cells, and includes HOX4 protein and NF-Ya protein .

"HOX4(Homeobox-leucine zipper protein, 서열번호 3)"는 전사인자 그룹으로 배아 형성에 중요한 역할을 한다. 전사인자는 줄기세포에서 유전자 발현 및 분화의 조절에 중요한 역할을 하며, 특정 유전자 표적에 대한 결합을 통하여 다양한 세포 과정을 전환한다. "HOX4 (Homeobox-leucine zipper protein, SEQ ID NO: 3)" is a transcription factor group and plays an important role in embryo formation. Transcription factors play an important role in the regulation of gene expression and differentiation in stem cells, and they convert various cellular processes through binding to specific gene targets.

"NF-Ya(Nuclear transcription factor Y subunit alpha, 서열번호 4)"는 CCAAT 프로모터 유전자의 주요한 전사 활성인자이며, p-53 의존성 전사 억제 메커니즘을 통해서 세포 스트레스에 의하여 세포 주기를 조절한다."NF-Ya (Nuclear transcription factor Y subunit alpha, SEQ ID NO: 4)" is a major transcriptional activator of the CCAAT promoter gene and regulates cell cycle by cell stress through a p-53 dependent transcriptional repression mechanism.

본 발명에서 사용되는 "세포 침투성 펩타이드(Cell-Penetrating Peptide; CPP)"는 양전하(+)를 띠는 펩타이드로서, (a) LMWP(Low Molecular Weight Protamine), (b) TAT(Trans Activator of Transcription)(서열번호 5), (c) 페너트라틴(penetratin)(서열번호 6), (d) 폴리아르기닌(polyarginine, 아르기닌 6개 이상), (e) 폴리라이신(polylysine, 라이신 6개 이상), (f) 프로타민(protamine) 절편, (g) 안테나페디아(antennapedia, ANTP) 및 (h)히스티딘, 아르기닌, 라이신 또는 이들의 혼합물을 70% 이상 함유하는 올리고펩타이드로 구성된 군에서 선택되는 것을 특징으로 할 수 있다. 또한 상기 세포 투과성 펩타이드(CPP)는 단백질 형질도입 부위(Protein Transduction Domain; PTD)를 포함하고 있으며, 상기 세포 투과성 펩타이드는 세포 투과성 단백질일 수도 있다."Cell-Penetrating Peptide (CPP)" used in the present invention is a peptide having a positive charge (+), which is (a) Low Molecular Weight Protamine (LMWP), (b) Trans Activator of Transcription (TAT) (D) polyarginine (at least 6 arginines), (e) polylysine (at least 6 lysines), (c) an oligopeptide comprising at least 70% of at least one polypeptide selected from the group consisting of f) protamine fragment, (g) antennapedia (ANTP) and (h) histidine, arginine, lysine or a mixture thereof. have. Also, the cell permeable peptide (CPP) includes a protein transduction domain (PTD), and the cell permeable peptide may be a cell permeable protein.

대부분의 세포내 약물 전달 기법은 세포 표면의 리셉터를 이용한 엔도사이토시스(endocytosis)에 의존하고 있으나 상기 펩타이드들은 기존의 세포내 물질 수송기전의 하나인 엔도사이토시스와는 전혀 다르게 (+) Charge를 띠고 있으므로 (-) Charge인 세포막의 전하에 의존하여 부착, 전달되는 과정을 거치며 세포내로 이동시키고자 하는 물질들을 직접적으로 단시간 내에 효율적인 도입을 시킬 수 있다는 장점을 가지고 있다. Most intracellular drug delivery techniques rely on endocytosis using cell surface receptors, but the peptides are (+) Charge differently from endocytosis, which is one of the pre- It has the advantage that it can directly introduce the substances to be transferred into the cell through a process of attaching and transferring depending on the charge of the cell membrane which is a (-) charge, in a short time.

본 발명에서 "줄기세포 재생"이란 줄기세포의 분화촉진능, 세포 사멸 억제능, 줄기세포의 증식능 향상, 줄기세포의 기능성 유지 및 스트레스에 의해 억제된 줄기세포의 기능성 회복을 포괄하는 것으로 정의한다.In the present invention, "stem cell regeneration" is defined as encompassing the function of promoting differentiation of stem cells, inhibiting apoptosis, enhancing the proliferative capacity of stem cells, maintaining the function of stem cells and restoring the function of stem cells inhibited by stress.

본 발명은 일 관점에서 항산화 단백질 또는 증식 향상능을 가지는 단백질의 아미노 말단에 세포 침투성 펩타이드가 결합된 줄기세포 재생 강화용 세포 침투성 융합 단백질에 관한 것이다.In one aspect, the present invention relates to a cell-penetrating fusion protein for enhancing stem cell regeneration wherein a cell permeable peptide is bound to an amino terminal of an antioxidant protein or a protein having a proliferation-improving ability.

본 발명은 다른 관점에서 항산화 단백질과 증식 향상능을 가지는 단백질과의 융합 단백질의 아미노 말단에 세포 침투성 펩타이드가 결합되어 있는 줄기세포 재생 또는 증식능 향상용 세포 침투성 융합 단백질에 관한 것이다.In another aspect, the present invention relates to a cell-penetrating fusion protein for enhancing stem cell regeneration or proliferation ability, wherein a cell permeability peptide is bound to the amino terminal of a fusion protein of an antioxidant protein and a protein having a proliferation-improving ability.

본 발명에 따른 상기 줄기세포능 증진용 세포 투과성 융합 단백질은 화학적 또는 생물학적으로 융합할 수 있으나, 부산물 등이 적게 발생하는 생물학적으로 융합시키는 것이 바람직하다. 화학적 융합방법은 상기 전사인자 단백질을 가교제로서 1,4-비스-말레이미도부탄(1,4-bis-maleimidobutane, BMB), 1,11-비스-말레이미도테트라에틸렌글리콜(1,11-bis-maleimidotetraethyleneglycol, BM[PEO]4), 1-에틸-3-[3-디메틸 아미노프로필] 카보디이미드 하이드로클로라이드(1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide hydrochloride, EDC), 숙시니미딜-4-[N-말레이미도메틸시클로헥산-1-카복시-[6-아미도카프로에이트]](succinimidyl-4-[N-maleimidomethyl cyclohexane-1-carboxy-[6-amidocaproate]], SMCC) 및 그의 설폰화염(sulfo-SMCC), 숙시미딜 6-[3-(2-피리딜디티오)-로피오나미도] 헥사노에이트](succimidyl 6-[3-(2-pyridyldithio)-ropionamido] hexanoate, SPDP) 및 그의 설폰화염(sulfo-SPDP), m-말레이미도벤조일-N-하이드로시숙시니미드 에스터(m-maleimidobenzoyl-N-hydroxysuccinimide ester, MBS) 및 그의 설폰화염(sulfo-MBS), 숙시미딜[4-(p-말레이미도페닐) 부틸레이트](succimidyl[4-(p-maleimidophenyl) butyrate], SMPB) 및 그의 설폰화염(sulfo-SMPB) 등을 이용하여 아미노기와 세포내 투과 펩타이드와 S-S 결합을 유도하는 것인데, 단백질의 아미노기에 비선택적으로 펩타이드 결합이 이루어져 한 단백질에 다수의 펩타이드가 도입되는 부산물이 발생한다. 생물학적 방법에 의해서는 플라스미드 구성시 한 분자의 펩타이드가 도입되도록 설계되어 있으므로 부산물의 발생우려가 적다는 장점이 있다. The cell-permeable fusion protein for stem cell function-increasing according to the present invention may be chemically or biologically fused, but it is preferably biologically fused to produce less by-products. The chemical fusion method uses the transcription factor protein as a crosslinking agent, 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethylene glycol (1,11-bis- maleimidotetraethyleneglycol, BM [PEO] 4), 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), succinimidyl- 4- [N-maleimidomethylcyclohexane-1-carboxy- [6-amidocaproate]], SMCC) and Succinimidyl 6- [3- (2-pyridyldithio) -propionamido] hexanoate, succinimidyl 6- [3- (2-pyridyldithio) SPDP) and its sulfone-SPDP, m-maleimidobenzoyl-N-hydroxysuccinimide ester, MBS and sulfo-MBS, [4- (p-maleimido (SMPB) and sulfo-SMPB (sulfo-SMPB) to induce the SS bond with the amino group and the intracellular permeable peptide, And a by-product in which a plurality of peptides are introduced into one protein is generated. Biological methods are designed to introduce one molecule of peptide in plasmid construction, so there is less concern about the occurrence of byproducts.

본 발명에 있어서, 상기 세포 침투성 펩타이드에 골조직 분화기능성 전사인자 단백질이 추가로 결합된 줄기세포 재생 강화용 세포 침투성 융합 단백질인 것을 특징으로 할 수 있다.In the present invention, the cell permeable peptide may further be a cell-penetrating fusion protein for enhancing stem cell regeneration in which a bone-differentiation-functioning transcription factor protein is further bound.

본 발명에서 "골조직 분화기능성 전사인자 단백질"은 중간엽 줄기세포에서 골격세포를 형성하는데 핵심적인 역할을 하는 전사인자를 뜻하며, TAZ, Runx2, LMP-1 및 이들의 유도체를 포함한다.In the present invention, "osteogenic differentiation-functioning transcription factor protein" refers to a transcription factor that plays a key role in forming skeletal cells in mesenchymal stem cells, and includes TAZ, Runx2, LMP-1 and derivatives thereof.

"TAZ (Tafazzin, 서열번호 7 또는 서열번호 8)"는 지방세포로 분화시키는 전사인자인 PPAR gamma를 억제하고, 중간엽 줄기세포에서 조골세포로 분화시키는 결정적 전사인자인 Runx2를 활성화시킬 수 있다. 또한 본 발명에서 TAZ는 상기 TAZ 단백질을 코딩하는 유전자를 의미할 수도 있다. "TAZ (Tafazzin, SEQ ID NO: 7 or SEQ ID NO: 8)" can activate Runx2, a crucial transcription factor that inhibits PPAR gamma, a transcription factor that differentiates into adipocytes, and differentiates into osteoblasts in mesenchymal stem cells. In the present invention, TAZ may also be a gene encoding the TAZ protein.

"Runx2 (Runx Domain transcription factor 2, 서열번호 9 또는 10)"는 중간엽 줄기세포에서 조골세포와 지방세포로 분화되는 결정적 전사인자이다. 이들은 중간엽 줄기세포들로 하여금 뼈를 만드는 조골세포 또는 지방세포로 차별화 하도록 유도한다. Runx2는 자체적으로도 외부에서 주입시 뼈로의 이행이 관찰된 단백질이며, 이 외에 여러 전사단백질들이 세포의 골조직으로의 분화를 촉진시키는 것으로 알려져 있다. "Runx2 (Runx Domain transcription factor 2, SEQ ID NO: 9 or 10)" is a crucial transcription factor that differentiates into osteoblasts and adipocytes in mesenchymal stem cells. They induce mesenchymal stem cells to differentiate into osteoblasts or adipocytes that make bone. Runx2 itself is a protein that has been observed to migrate from the outside to the bone at the time of injection, and various transcriptional proteins are known to promote cell differentiation into bone tissue.

"LMP-1 (LIM Mineralization protein-1, 서열번호 11 또는 12) 역시 세포내에서 골조직으로의 분화를 촉진시킨다. 특히 본 발명자들은 이 단백질 중 특정의 아미노산 서열 (AADPPRYTFAPSVSLNKTARPPGAPPPADSAPQQNG, ADPPRYTFAP, KPQKASAPAADPPRYTFAP)이 골조직분화와 직접관련이 있음을 확인한 바 있다. In particular, the present inventors have found that a specific amino acid sequence (AADPPRYTFAPSVSLNKTARPPGGAPPPADSAPQQNG, ADPPRYTFAP, KPQKASAPAADPPRYTFAP) of this protein is involved in bone differentiation (LMP-1, And that it is directly related to

본 발명은 또 다른 관점에서 세포 침투성 펩타이드 형질 도입 부위를 coding 하는 cDNA; 항산화 단백질 형질 도입 부위를 coding 하는 cDNA 및/또는 증식 향상능을 가지는 단백질 형질 도입 부위를 coding 하는 cDNA; 골조직 분화기능성 전사인자 단백질을 coding 하는 cDNA; 및 6개의 히스티딘에 대한 cDNA를 포함하고 있으며, 줄기세포 재생 강화능을 가지는 융합 단백질의 발현 벡터에 관한 것이다.In another aspect, the present invention relates to a cDNA encoding a cell penetrating peptide transduction site; A cDNA coding for an antioxidant protein transduction site and / or a cDNA coding for a protein transduction site having a proliferation improving ability; CDNA coding for a bone-differentiation functional transcription factor protein; And six histidine cDNAs, and relates to an expression vector of a fusion protein having stem cell regeneration-enhancing ability.

상기 벡터는 통상적인 TA 벡터 등을 사용할 수 있고, 상기 벡터의 발현은 T7 프로모터와 LacO-오퍼레이터의 조절하에 있다.The vector may be a conventional TA vector or the like, and expression of the vector is under the control of a T7 promoter and a LacO-operator.

본 발명은 또 다른 관점에서 세포 침투성 펩타이드 형질 도입 부위를 coding 하는 cDNA; 항산화 단백질 형질 도입 부위를 coding 하는 cDNA 및/또는 증식 향상능을 가지는 단백질 형질 도입 부위를 coding 하는 cDNA; 골조직 분화기능성 전사인자 단백질을 coding 하는 cDNA; 및 6개의 히스티딘에 대한 cDNA를 포함하고 있으며, 줄기세포 재생 강화능을 가지는 융합 단백질의 발현 벡터로 형질전환된 재조합 미생물에 관한 것이다.In another aspect, the present invention relates to a cDNA encoding a cell penetrating peptide transduction site; A cDNA coding for an antioxidant protein transduction site and / or a cDNA coding for a protein transduction site having a proliferation improving ability; CDNA coding for a bone-differentiation functional transcription factor protein; And a cDNA for six histidines, and a recombinant microorganism transformed with an expression vector of a fusion protein having stem cell regeneration-enhancing ability.

상기 형질전환용 미생물은 통상적인 것을 사용할 수 있고, E· Coli 등을 예시할 수 있다.As the microorganism for transformation, a conventional one can be used, and E. Coli and the like can be exemplified.

본 발명은 또 다른 관점에서 세포 침투성 펩타이드 형질 도입 부위를 coding 하는 cDNA; 항산화 단백질 형질 도입 부위를 coding 하는 cDNA 및/또는 증식 향상능을 가지는 단백질 형질 도입 부위를 coding 하는 cDNA; 골조직 분화기능성 전사인자 단백질을 coding 하는 cDNA; 및 6개의 히스티딘에 대한 cDNA를 포함하고 있으며, 줄기세포 재생 강화능을 가지는 융합 단백질의 발현 벡터로 형질전환된 재조합 미생물을 배양하여 줄기세포 재생 강화능을 가지는 융합 단백질을 발현시키는 단계; 및 상기 발현된 융합 회수하는 단계를 포함하는 줄기세포 재생 강화능을 가지는 융합 단백질의 제조방법에 관한 것이다.In another aspect, the present invention relates to a cDNA encoding a cell penetrating peptide transduction site; A cDNA coding for an antioxidant protein transduction site and / or a cDNA coding for a protein transduction site having a proliferation improving ability; CDNA coding for a bone-differentiation functional transcription factor protein; And expressing a fusion protein having a stem cell regeneration-enhancing ability by culturing a recombinant microorganism transformed with an expression vector of a fusion protein having a stem cell regeneration-enhancing ability, the cDNA comprising 6 histidines; And recovering the expressed fusion. The present invention also relates to a method for producing a fusion protein having stem cell regeneration enhancing ability.

본 발명은 또 다른 관점에서 세포 침투성 펩타이드 형질 도입 부위를 coding 하는 cDNA; 항산화 단백질 형질 도입 부위를 coding 하는 cDNA 및/또는 증식 향상능을 가지는 단백질 형질 도입 부위를 coding 하는 cDNA; 골조직 분화기능성 전사인자 단백질을 coding 하는 cDNA; 및 6개의 히스티딘에 대한 cDNA를 포함하고 있으며, 줄기세포 재생 강화능을 가지는 융합 단백질의 발현 벡터로 형질전환된 재조합 미생물을 배양하여 줄기세포 재생 강화능을 가지는 융합 단백질을 발현시키는 단계; 및 상기 발현된 융합 회수하는 단계를 포함하는 줄기세포 재생 강화능을 가지는 융합 단백질의 제조방법에 의해 제조된 융합 단백질을 줄기세포에 도입하는 것을 특징으로 하는 줄기세포의 재생방법에 관한 것이다.In another aspect, the present invention relates to a cDNA encoding a cell penetrating peptide transduction site; A cDNA coding for an antioxidant protein transduction site and / or a cDNA coding for a protein transduction site having a proliferation improving ability; CDNA coding for a bone-differentiation functional transcription factor protein; And expressing a fusion protein having a stem cell regeneration-enhancing ability by culturing a recombinant microorganism transformed with an expression vector of a fusion protein having a stem cell regeneration-enhancing ability, the cDNA comprising 6 histidines; And a step of introducing the fusion protein produced by the method for producing a fusion protein having a stem cell regeneration enhancing ability into a stem cell.

본 발명은 또 다른 관점에서 항산화 단백질 또는 증식 향상능을 가지는 단백질과 골조직 분화기능성 전사인자 단백질과의 융합 단백질의 말단에 세포 침투성 펩타이드가 결합되어 있는 골형성 촉진용 세포 침투성 융합 단백질에 관한 것이다.In another aspect, the present invention relates to a cell permeable fusion protein for promoting bone formation, wherein a cell permeable peptide is bound to the end of a fusion protein of a protein having antioxidant protein or proliferation improving ability and a bone morphogenic differentiation functional transcription factor protein.

본 발명은 또 다른 관점에서 항산화 단백질 또는 증식 향상능을 가지는 단백질과 골조직 분화기능성 전사인자 단백질과의 융합 단백질의 말단에 세포 침투성 펩타이드가 결합되어 있는 골형성 촉진용 세포 침투성 융합 단백질을 유효성분으로 포함하는 골질환 치료용 약학적 조성물에 관한 것이다.In another aspect of the present invention, there is provided a pharmaceutical composition comprising, as an active ingredient, a cell permeable fusion protein for promoting bone formation, wherein a cell permeable peptide is bound to the end of a fusion protein of a protein having antioxidant protein or proliferation improving ability and a bone morphogenic differentiation functional transcription factor protein To a pharmaceutical composition for the treatment of bone diseases.

본 발명에 있어서, 상기 골질환은 골다공증, 골형성 부전증, 치주질환 및 골절로 구성된 군에서 선택된 어느 하나인 것을 특징으로 한다.In the present invention, the bone disease is any one selected from the group consisting of osteoporosis, osteogenesis imperfecta, periodontal disease, and fracture.

상기 골질환 치료용 약학 조성물은 경구 투여 또는 비경구 투여(근육내, 피하, 정맥내, 좌약 등)에 의해 투여될 수 있다. 적당한 투여량은 환자의 상태, 예를 들어, 연령 및 증상의 정도 등에 따라 적절히 조절할 수 있지만, 통상 성인의 경우 100~500mg의 용량 범위에서 일반적으로 일회 또는 수 회로 나누어 1일당 100~1,000mg의 양으로, 바람직하게는 1일 300~1,000mg의 양으로 투여되는 것이 바람직하다.The pharmaceutical composition for treating bone diseases can be administered by oral administration or parenteral administration (intramuscular, subcutaneous, intravenous, suppository, etc.). The appropriate dosage can be appropriately adjusted depending on the condition of the patient, for example, the age and the degree of symptoms, but is usually in the range of 100-500 mg for adults, generally divided into once or several times a dose of 100-1,000 mg per day , Preferably in an amount of 300 to 1,000 mg per day.

본 발명에 따른 상기 골질환 치료용 약학 조성물을 경구용 제제로서 조제하는 경우에는 부형제, 필요에 따라서 결합제, 붕괴제, 활택제, 착색제, 교미교취제 등을 가한 후, 통상적인 방법에 따라 정제, 피복 정제, 과립제, 캡슐제 등으로 제조할 수 있다.When the pharmaceutical composition for treating bone diseases according to the present invention is prepared as an oral preparation, an excipient, if necessary, a binder, a disintegrant, a lubricant, a colorant, a mating agent or the like is added, Tablets, granules, capsules and the like.

상기 골질환 치료용 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 젖당, 옥수수 전분, 백당, 포도당, 솔비트, 결정 셀룰로스, 만니톨, 자일리톨, 에리스리톨, 말티톨, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 사용할 수 있고, 결합제로는 폴리비닐알콜, 폴리비닐에테르, 에틸셀룰로스, 메틸셀룰로스, 아라비아 고무, 젤라틴, 셸락(shellac), 하이드록시프로필셀룰로스, 하이드록시프로필스타치, 폴리비닐피리돈 등을 사용할 수 있다. 그리고, 붕괴제로는 전분, 한천, 젤라틴 분말, 결정 셀룰로스, 탄산칼슘, 탄산수소나트륨, 구연산칼슘, 덱스트란, 펙틴 등이, 활택제로는 스테아린산 마그네슘, 활석, 폴리에틸렌글리콜, 실리카, 경화 식물유 등을 사용할 수 있다. 또한, 착색제로는 의약품에 첨가하는 것이 허가된 것을 사용할 수 있고, 교미교취제로 코코아 분말, 박하뇌, 방향산, 박하유, 용뇌, 계피 분말 등을 사용할 수 있다. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition for treating bone diseases include lactose, corn starch, white sugar, glucose, sorbitol, crystalline cellulose, mannitol, xylitol, erythritol, maltitol, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose , Methyl cellulose, gum arabic, gelatin, shellac, hydroxypropylcellulose, hydroxypropyl starch, polyvinylpyridone and the like. Examples of the disintegrator include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, calcium citrate, dextran and pectin. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, . As the coloring agent, it is possible to use those which are allowed to be added to medicines, and as a mating agent, cocoa powder, peppermint, oriental acid, peppermint oil, cerebrospinal fluid, cinnamon powder and the like can be used.

또한 본 발명에 따른 골질환 치료용 약학 조성물을 주사제로 조제하는 경우에는 필요에 따라, pH 조절제, 완충제, 안정화제, 보존제 등을 첨가하고, 통상적인 방법에 따라 피하, 근육내, 정맥 주사제로 조제할 수 있다.When a pharmaceutical composition for treating bone diseases according to the present invention is prepared by injection, a pH adjusting agent, a buffering agent, a stabilizing agent, a preservative, etc. may be added as needed. Subcutaneous, intramuscular, intravenous can do.

본 발명에 따른 골질환 치료용 약학 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 (intracerebroventricular) 주사에 의해 투여될 수 있다.The pharmaceutical composition for treating bone diseases according to the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

본 발명은 또 다른 관점에서 항산화 단백질 또는 증식 향상능을 가지는 단백질과 골조직 분화기능성 전사인자 단백질과의 융합 단백질의 말단에 세포 침투성 펩타이드가 결합되어 있는 골형성 촉진용 세포 침투성 융합 단백질을 유효성분으로 하고, 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 것을 특징으로 하는 골질환 치료용 건강기능성 식품에 관한 것이다.In another aspect, the present invention provides, as an active ingredient, a cell permeable fusion protein for promoting bone formation, wherein a cell permeable peptide is bound to the end of a fusion protein of a protein having antioxidant protein or proliferation improving ability and a bone morphogenic differentiation functional transcription factor protein , And a food-acceptable food-aid additive. The present invention also relates to a health functional food for treating bone diseases.

"기능성 식품"이란, 일반 식품에 본 발명의 골형성 촉진용 세포투과성 융합 단백질(CPP-TAZ)을 첨가함으로써 일반 식품의 기능성을 향상시킨 식품을 의미한다. The term "functional food" means a food having improved functionality of a general food by adding the cell permeable fusion protein (CPP-TAZ) for promoting bone formation to the general food.

본 발명에 따른 골질환용 건강 기능성 식품은 식품학적으로 허용 가능한 식품보조 첨가제로 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명에 따른 골질환용 건강 기능성 식품은 천연 과일 쥬스, 과일 쥬스 음료 및 야채 음료 제조를 위한 과육 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 특별히 제한되는 것은 아니지만, 본 발명의 골형성 촉진용 세포투과성 융합 단백질 100중량부 당 0.01~20중량부의 범위에서 선택되는 것이 일반적이다.The health functional food for bone diseases according to the present invention is a food-acceptable food-aid additive which is used as a nutritional supplement, a vitamin, a mineral (electrolyte), a flavor such as a synthetic flavor and a natural flavor, a coloring agent, ), Pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. In addition, the health functional food for bone diseases according to the present invention may contain natural fruit juice, fruit juice drink, and pulp for producing vegetable beverages. These components may be used independently or in combination. The ratio of such additives is not particularly limited, but is generally selected in the range of 0.01 to 20 parts by weight per 100 parts by weight of the cell permeable fusion protein for promoting bone formation of the present invention.

이하, 하기 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to the following examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

특히, 본 실시예에서는 LMWP와 Thioredoxin 또는 SOD와의 결합에 의한 융합 단백질을 줄기세포에 처리하는 경우, 줄기세포능이 증진된다는 것을 예시하였으나, 통상의 세포 투과성 펩타이드와 항산화 단백질이 결합된 융합 단백질에 있어서 줄기세포능이 증진된다는 것 역시 당업자에게 자명하다 할 것인 바, 본 발명의 범위에 속한다 할 것이다.
Particularly, in the present embodiment, it has been exemplified that when stem cells are treated with a fusion protein by binding LMWP with Thioredoxin or SOD, stem cell performance is enhanced. However, in a fusion protein in which a normal cell permeable peptide and an antioxidant protein are combined, It will also be apparent to those skilled in the art that cell viability is enhanced, which is within the scope of the present invention.

재조합 Recombination LMWPLMWP -- ThoredoxinThoredoxin -1 융합 단백질의 제조 방법 및 세포 내 투과능력 확인-1 fusion protein and its intracellular permeability

LMWP 펩타이드를 합성한 후, thioredoxin (1 mg/ml in PBS)를 SMCC (Pierce)를 사용하여 conjugation을 수행하였다. Conjugate된 LMWP-thioredoxin 복합체는 heparin-agarose bead (Sigma)를 사용하여 정제하였다.LMWP peptides were synthesized and thioredoxin (1 mg / ml in PBS) was conjugated using SMCC (Pierce). Conjugated LMWP-thioredoxin complexes were purified using heparin-agarose beads (Sigma).

모델 세포주인 C2C12 세포주(ATCC)에 혈청이 들어가지 않은 배지를 첨가하여 24시간 동안 배양((95% air, 5% CO2, 37℃)하였다. 그런 후에 LMWP-Trx-1(WT과 C3134S) 복합체들을 모델 세포주 (mouse C2C12)에 혈청이 없는 배지에 30min 동안 처리하여 배양 후 conjugation시키지 않은 Thioredoxin-1과 비교실험을 수행하였다. 그 결과 Thioredoxin-1만 처리한 대조군과 비교하였을 때 세포내 투과 능력이 뛰어난 것을 확인하였으며 C3134S 복합체에 비해서 WT 복합체의 세포투과능이 더 뛰어난 것을 확인하였다 (도 1).
(WT and C3134S) complexes were added to the C2C12 cell line (ATCC), a model cell line, and cultured for 24 hours (95% air, 5% Were compared with Thioredoxin-1, which was not conjugated after incubation for 30 min in a serum-free medium (mouse C2C12). As a result, the intracellular permeability And the cell permeability of the WT complex was superior to that of the C3134S complex (Fig. 1).

재조합 Recombination LMWPLMWP -- ThoredoxinThoredoxin -1 융합 단백질의 줄기세포 분화 -1 Fusion Protein Stem Cell Differentiation 촉진능Acceleration ability 확인 Confirm

LMWP-Thioredoxin-1 융합단백질이 줄기세포(Lonza)로부터 근육세포로의 분화에 미치는 영향을 확인하기 위해서, LMWP-Thioredoxin-1-WT 복합체를 줄기세포에 농도별로 처리하여 일주일동안 배양 후((95% air, 5% CO2, 37℃), Western blotting 기법을 사용하여 근육세포 분화 marker인 MYH, Myogenin, MyoD의 단백질 발현양을 확인하였다. 본 실험에서는 LMWP와 conjugation을 시키지 않은 Thioredoxin-1을 대조군으로 사용하였다. 그 결과 LMWP-Thioredoxin-1-WT 복합체를 처리하였을 때 농도 의존적으로 근육세포로의 분화가 증가하는 것을 확인하였다 (도 2).
To determine the effect of LMWP-Thioredoxin-1 fusion protein on the differentiation of stem cells (Lonza) into muscle cells, LMWP-Thioredoxin-1-WT complex was treated at different concentrations in stem cells for one week MYH, Myogenin, and MyoD, which are the differentiation markers of muscle cells, were identified by Western blotting. Thioredoxin-1, which was not conjugated with LMWP, was used as a control As a result, it was confirmed that when the LMWP-Thioredoxin-1-WT complex was treated, the differentiation into muscle cells was increased in a concentration-dependent manner (FIG. 2).

재조합 Recombination LMWPLMWP -- SODSOD 융합 단백질의 제조방법 및 세포사멸 억제 능력 확인 Confirmation of fusion protein production method and cytotoxicity

LMWP 펩타이드를 합성한 후, SOD (Sigma, 1 mg/ml in PBS)를 SMCC (Pierce)를 사용하여 conjugation을 수행하였다. Conjugate된 LMWP-SOD 복합체는 heparin-agarose bead (Sigma)를 사용하여 정제하였다.LMWP peptides were synthesized and conjugated with SOD (Sigma, 1 mg / ml in PBS) using SMCC (Pierce). Conjugated LMWP-SOD complexes were purified using heparin-agarose beads (Sigma).

세포 투과 능력을 지닌 LMWP-SOD 융합단백질이 세포사멸에 어떠한 능력을 미치는 지 확인하였다. 6 well에 human dental pulp stem cell(primary culture)을 1X 10⁴개 seeding 후 24시간 동안 배양(95% air, 5% CO2, 37℃) 배양하였다. 그런 후 LMWP-SOD 복합체를 농도별로 처리하여 2시간 동안 배양한 다음 혈청이 없는 배지에 hydrogen peroxide를 낮은 농도(150 uM)로 2시간 동안 처리하여 인위적으로 세포 노화를 유발시키고 일반 배지로 교체 후 3일 동안 배양하였다. 세포의 노화 정도는 세포의 모양변화 (도 3A) 및 senesence associated beta-galactosidase 염색 (도 3B, 도 3C)을 통해 확인하였다. 그 결과, LMWP-SOD를 전처리한 실험군에서 flatten한 세포의 모양이 부분적으로 회복되는 것을 확인하였고, SA-beta-gal 염색이 확연하게 줄어든 것을 확인할 수 있었다.
We confirmed the ability of LMWP-SOD fusion protein with cell permeability to kill cells. Human dental pulp stem cells (primary culture) were seeded in 6 wells and cultured for 24 hours (95% air, 5% CO2, 37 ℃). Then, the LMWP-SOD complex was cultured for 2 hours and then treated with hydrogen peroxide at a low concentration (150 uM) for 2 hours in serum-free medium to induce cell aging artificially. Lt; / RTI > The degree of senescence of the cells was confirmed by changes in the shape of cells (Fig. 3A) and senesence associated beta-galactosidase staining (Fig. 3B, Fig. 3C). As a result, it was confirmed that the shape of flattened cells was partially restored in the LMWP-SOD pretreated group, and SA-beta-gal staining was significantly reduced.

재조합 Recombination LMWPLMWP -- SODSOD 융합 단백질의 줄기세포 기능성 유지 및 관여 단백질 확인 Maintain stem cell function of fusion proteins and identify proteins involved

세포 투과 능력을 지닌 LMWP-SOD 복합체가 줄기세포 기능성에 어떠한 능력을 미치는 지 확인하였다. 100mm dish에 human dental pulp stem cell을 1X 106 개seeding 후 24시간 동안 배양하였다. 그런 후 LMWP-SOD 복합체를 농도별로 처리하여 2시간 동안 배양(95% air, 5% CO2, 37℃)한 다음, 혈청이 없는 배지에 hydrogen peroxide를 낮은 농도(150 uM)로 2시간 동안 처리하여 인위적으로 세포 스트레스를 유발시키고 일반 배지로 교체 후 3일 동안 배양하였다. 세포의 노화에 관여한다는 p53 유전자및 단백질고 그 하위에서 p53에 의해 활성이 조절되는 p21 유전자 및 단백질의 변화를 RT-PCR (도 4A, 도 4B)와 Western blotting 기법을 (도 4C, 도 4D) 통해 확인하였다. 그 결과 LMWP-SOD를 전처리한 실험군에서 p53-p21 유전자의 발현 수준도 감소하였으며 단백질의 발현전도도 현저히 감소하는 것을 확인할 수 있었다. 본 실험을 통해서 독성이 없으며 세포내로 특정 단백질을 효율적으로 전달할 수 있는 기능을 보유한 생리활성 펩타이드의 도입으로 노화에 필요한 SOD 단백질의 발현을 증가시켜 과한화수소에 의한 세포 수준의 스트레스를 억제한다는 것을 확인하였으며, 이 과정에서 p53-p21 신호전달계가 관여한다는 것을 확인하였다.
The ability of the LMWP-SOD complex with cell permeability to regulate stem cell function was tested. Human dental pulp stem cells were seeded in 1x10 6 dish and cultured for 24 hours. Then, the LMWP-SOD complex was cultured for 2 hours (95% air, 5% CO 2, 37 ° C) and treated with hydrogen peroxide at a low concentration (150 μM) for 2 hours Cell stress was induced artificially and cultured for 3 days after replacing with normal medium. 4A and 4B) and Western blotting (Figs. 4C and 4D), and p21 gene and protein, which are involved in cell senescence, and p21 gene and protein whose activity is regulated by p53, Respectively. As a result, the expression level of p53-p21 gene was decreased in the experimental group pretreated with LMWP-SOD, and the expression of protein was significantly decreased. In this experiment, it was confirmed that the introduction of physiologically active peptides having no toxicity and the ability to efficiently transfer specific proteins into cells increased the expression of SOD protein required for aging and suppressed cell-level stress due to excess hydrogen , Suggesting that the p53-p21 signaling pathway is involved in this process.

재조합 Recombination LMWPLMWP -- SODSOD 융합 단백질에 의한 스트레스 억제된 줄기세포의 기능성 회복 Functional restoration of stress-restricted stem cells by fusion protein

줄기세포는 주변상황 변화등에 의한 스트레스를 받게 되면 그 줄기세포의 기능성이 상실되게 된다. 본 실험에서도 이런 관점에 초점을 두어 LMWP-SOD 복합체가 세포내로 투과되어 노화된 줄기세포(Lonza)의 기능성을 회복할 수 있는지 여부를 Alizarin red S 염색을 통해 석회화 정도를 측정하였다. LMWP-SOD 복합체를 과산화수소 2시간 전에 처리 후 14일 동안 배양(95% air, 5% CO2, 37℃)하였다. 그런 후 Alizarin red S 염색을 하여 석회화 정도를 측정한 결과 SOD만 처리한 군이나 대조군과 비교하였을 때 LMWP-SOD를 처리한 군에서 석회화가 회복 되는 것을 확인하였다 (도 5A, 도 5B). 이런 줄기세포의 기능성 회복효과를 확인하기 위해서 경조족 분화 유전자인 ALP, type I collagen, osteopontin mRNA 수준을 확인하였다. 그 결과, 그림 A-B와 유사한 패턴으로 LMWP-SOD를 처리한 군에서 이들 유전자의 발현양이 회복되는 것을 확인하였다 (도 5C, 도 5D). When stem cells are subjected to stress due to changes in their surroundings, the function of the stem cells is lost. In this experiment, too, the degree of calcification was measured by Alizarin red S staining to determine whether the LMWP-SOD complex could penetrate into the cell and restore the function of aged stem cells (Lonza). The LMWP-SOD complex was treated with hydrogen peroxide for 2 hours before culturing (95% air, 5% CO2, 37 ° C) for 14 days. After calcination with Alizarin red S, calcification was restored in the LMWP-SOD treated group compared with the SOD-treated group or the control group (FIGS. 5A and 5B). In order to confirm the functional recovery of these stem cells, the levels of ALP, type I collagen and osteopontin mRNA were examined. As a result, it was confirmed that the expression level of these genes was restored in the group treated with LMWP-SOD in a pattern similar to that of FIG. A-B (FIG. 5C, FIG. 5D).

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Seoul National University Industry Foundation <120> Cell Permeable Fusion Protein for Strengthening Regenerative Potential of Stem Cells <130> P11-B104 <160> 12 <170> KopatentIn 2.0 <210> 1 <211> 153 <212> PRT <213> SOD-HUMAN <400> 1 Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly 1 5 10 15 Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp 20 25 30 Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His 35 40 45 Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe 50 55 60 Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His 65 70 75 80 Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp 85 90 95 Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile 100 105 110 Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys 115 120 125 Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu 130 135 140 Ala Cys Gly Val Ile Gly Ile Ala Gln 145 150 <210> 2 <211> 103 <212> PRT <213> Thioredoxin-HUMAN <400> 2 Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala 1 5 10 15 Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly 20 25 30 Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys Tyr 35 40 45 Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp Val 50 55 60 Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe Lys 65 70 75 80 Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu 85 90 95 Glu Ala Thr Ile Asn Glu Leu 100 <210> 3 <211> 277 <212> PRT <213> HOX4 <400> 3 Met Lys Arg Pro Gly Gly Ala Gly Gly Gly Gly Gly Ser Pro Ser Leu 1 5 10 15 Val Thr Met Ala Asn Ser Ser Asp Asp Gly Tyr Gly Gly Val Gly Met 20 25 30 Glu Ala Glu Gly Asp Val Glu Glu Glu Met Met Ala Cys Gly Gly Gly 35 40 45 Gly Glu Lys Lys Arg Arg Leu Ser Val Glu Gln Val Arg Ala Leu Glu 50 55 60 Arg Ser Phe Glu Val Glu Asn Lys Leu Glu Pro Glu Arg Lys Ala Arg 65 70 75 80 Leu Ala Arg Asp Leu Gly Leu Gln Pro Arg Gln Val Ala Val Trp Phe 85 90 95 Gln Asn Arg Arg Ala Arg Trp Lys Thr Lys Gln Leu Glu Arg Asp Tyr 100 105 110 Ala Ala Leu Arg His Ser Tyr Asp Ser Leu Arg Leu Asp His Asp Ala 115 120 125 Leu Arg Arg Asp Lys Asp Ala Leu Leu Ala Glu Ile Lys Glu Leu Lys 130 135 140 Ala Lys Leu Gly Asp Glu Glu Ala Ala Ala Ser Phe Thr Ser Val Lys 145 150 155 160 Glu Glu Pro Ala Ala Ser Asp Gly Pro Pro Ala Ala Gly Phe Gly Ser 165 170 175 Ser Asp Ser Asp Ser Ser Ala Val Leu Asn Asp Val Asp Ala Ala Gly 180 185 190 Ala Ala Pro Ala Ala Thr Asp Ala Leu Ala Pro Glu Ala Cys Thr Phe 195 200 205 Leu Gly Ala Pro Pro Ala Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala 210 215 220 Ala Ser His Glu Glu Val Phe Phe His Gly Asn Phe Leu Lys Val Glu 225 230 235 240 Glu Asp Glu Thr Gly Phe Leu Asp Asp Asp Glu Pro Cys Gly Gly Phe 245 250 255 Phe Ala Asp Asp Gln Pro Pro Pro Leu Ser Ser Trp Trp Ala Glu Pro 260 265 270 Thr Glu His Trp Asn 275 <210> 4 <211> 347 <212> PRT <213> NF-Ya <400> 4 Met Glu Gln Tyr Thr Ala Asn Ser Asn Ser Ser Thr Glu Gln Ile Val 1 5 10 15 Val Gln Ala Gly Gln Ile Gln Gln Gln Gln Gln Gly Gly Val Thr Ala 20 25 30 Val Gln Leu Gln Thr Glu Ala Gln Val Ala Ser Ala Ser Gly Gln Gln 35 40 45 Val Gln Thr Leu Gln Val Val Gln Gly Gln Pro Leu Met Val Gln Val 50 55 60 Ser Gly Gly Gln Leu Ile Thr Ser Thr Gly Gln Pro Ile Met Val Gln 65 70 75 80 Ala Val Pro Gly Gly Gln Gly Gln Thr Ile Met Gln Val Pro Val Ser 85 90 95 Gly Thr Gln Gly Leu Gln Gln Ile Gln Leu Val Pro Pro Gly Gln Ile 100 105 110 Gln Ile Gln Gly Gly Gln Ala Val Gln Val Gln Gly Gln Gln Gly Gln 115 120 125 Thr Gln Gln Ile Ile Ile Gln Gln Pro Gln Thr Ala Val Thr Ala Gly 130 135 140 Gln Thr Gln Thr Gln Gln Gln Ile Ala Val Gln Gly Gln Gln Val Ala 145 150 155 160 Gln Thr Ala Glu Gly Gln Thr Ile Val Tyr Gln Pro Val Asn Ala Asp 165 170 175 Gly Thr Ile Leu Gln Gln Val Thr Val Pro Val Ser Gly Met Ile Thr 180 185 190 Ile Pro Ala Ala Ser Leu Ala Gly Ala Gln Ile Val Gln Thr Gly Ala 195 200 205 Asn Thr Asn Thr Thr Ser Ser Gly Gln Gly Thr Val Thr Val Thr Leu 210 215 220 Pro Val Ala Gly Asn Val Val Asn Ser Gly Gly Met Val Met Met Val 225 230 235 240 Pro Gly Ala Gly Ser Val Pro Ala Ile Gln Arg Ile Pro Leu Pro Gly 245 250 255 Ala Glu Met Leu Glu Glu Glu Pro Leu Tyr Val Asn Ala Lys Gln Tyr 260 265 270 His Arg Ile Leu Lys Arg Arg Gln Ala Arg Ala Lys Leu Glu Ala Glu 275 280 285 Gly Lys Ile Pro Lys Glu Arg Arg Lys Tyr Leu His Glu Ser Arg His 290 295 300 Arg His Ala Met Ala Arg Lys Arg Gly Glu Gly Gly Arg Phe Phe Ser 305 310 315 320 Pro Lys Glu Lys Asp Ser Pro His Met Gln Asp Pro Asn Gln Ala Asp 325 330 335 Glu Glu Ala Met Thr Gln Ile Ile Arg Val Ser 340 345 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> TAT(Transactivator of Transcription Peptide from HIV type-1) <400> 5 Cys Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Cys 1 5 10 15 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Penetratin <400> 6 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 7 <211> 400 <212> PRT <213> TAZ-HUMAN <400> 7 Met Asn Pro Ala Ser Ala Pro Pro Pro Leu Pro Pro Pro Gly Gln Gln 1 5 10 15 Val Ile His Val Thr Gln Asp Leu Asp Thr Asp Leu Glu Ala Leu Phe 20 25 30 Asn Ser Val Met Asn Pro Lys Pro Ser Ser Trp Arg Lys Lys Ile Leu 35 40 45 Pro Glu Ser Phe Phe Lys Glu Pro Asp Ser Gly Ser His Ser Arg Gln 50 55 60 Ser Ser Thr Asp Ser Ser Gly Gly His Pro Gly Pro Arg Leu Ala Gly 65 70 75 80 Gly Ala Gln His Val Arg Ser His Ser Ser Pro Ala Ser Leu Gln Leu 85 90 95 Gly Thr Gly Ala Gly Ala Ala Gly Ser Pro Ala Gln Gln His Ala His 100 105 110 Leu Arg Gln Gln Ser Tyr Asp Val Thr Asp Glu Leu Pro Leu Pro Pro 115 120 125 Gly Trp Glu Met Thr Phe Thr Ala Thr Gly Gln Arg Tyr Phe Leu Asn 130 135 140 His Ile Glu Lys Ile Thr Thr Trp Gln Asp Pro Arg Lys Ala Met Asn 145 150 155 160 Gln Pro Leu Asn His Met Asn Leu His Pro Ala Val Ser Ser Thr Pro 165 170 175 Val Pro Gln Arg Ser Met Ala Val Ser Gln Pro Asn Leu Val Met Asn 180 185 190 His Gln His Gln Gln Gln Met Ala Pro Ser Thr Leu Ser Gln Gln Asn 195 200 205 His Pro Thr Gln Asn Pro Pro Ala Gly Leu Met Ser Met Pro Asn Ala 210 215 220 Leu Thr Thr Gln Gln Gln Gln Gln Gln Lys Leu Arg Leu Gln Arg Ile 225 230 235 240 Gln Met Glu Arg Glu Arg Ile Arg Met Arg Gln Glu Glu Leu Met Arg 245 250 255 Gln Glu Ala Ala Leu Cys Arg Gln Leu Pro Met Glu Ala Glu Thr Leu 260 265 270 Ala Pro Val Gln Ala Ala Val Asn Pro Pro Thr Met Thr Pro Asp Met 275 280 285 Arg Ser Ile Thr Asn Asn Ser Ser Asp Pro Phe Leu Asn Gly Gly Pro 290 295 300 Tyr His Ser Arg Glu Gln Ser Thr Asp Ser Gly Leu Gly Leu Gly Cys 305 310 315 320 Tyr Ser Val Pro Thr Thr Pro Glu Asp Phe Leu Ser Asn Val Asp Glu 325 330 335 Met Asp Thr Gly Glu Asn Ala Gly Gln Thr Pro Met Asn Ile Asn Pro 340 345 350 Gln Gln Thr Arg Phe Pro Asp Phe Leu Asp Cys Leu Pro Gly Thr Asn 355 360 365 Val Asp Leu Gly Thr Leu Glu Ser Glu Asp Leu Ile Pro Leu Phe Asn 370 375 380 Asp Val Glu Ser Ala Leu Asn Lys Ser Glu Pro Phe Leu Thr Trp Leu 385 390 395 400 <210> 8 <211> 395 <212> PRT <213> TAZ-MOUSE <400> 8 Met Asn Pro Ser Ser Val Pro His Pro Leu Pro Pro Pro Gly Gln Gln 1 5 10 15 Val Ile His Val Thr Gln Asp Leu Asp Thr Asp Leu Glu Ala Leu Phe 20 25 30 Asn Ser Val Met Asn Pro Lys Pro Ser Ser Trp Arg Lys Lys Ile Leu 35 40 45 Pro Glu Ser Phe Phe Lys Glu Pro Asp Ser Gly Ser His Ser Arg Gln 50 55 60 Ser Ser Thr Asp Ser Ser Gly Gly His Pro Gly Pro Arg Leu Ala Gly 65 70 75 80 Gly Ala Gln His Val Arg Ser His Ser Ser Pro Ala Ser Leu Gln Leu 85 90 95 Gly Thr Gly Ala Gly Ala Ala Gly Gly Pro Ala Gln Gln His Ala His 100 105 110 Leu Arg Gln Gln Ser Tyr Asp Val Thr Asp Glu Leu Pro Leu Pro Pro 115 120 125 Gly Trp Glu Met Thr Phe Thr Ala Thr Gly Gln Arg Tyr Phe Leu Asn 130 135 140 His Ile Glu Lys Ile Thr Thr Trp Gln Asp Pro Arg Lys Val Met Asn 145 150 155 160 Gln Pro Leu Asn His Val Asn Leu His Pro Ser Ile Thr Ser Thr Ser 165 170 175 Val Pro Gln Arg Ser Met Ala Val Ser Gln Pro Asn Leu Ala Met Asn 180 185 190 His Gln His Gln Gln Val Val Ala Thr Ser Leu Ser Pro Gln Asn His 195 200 205 Pro Thr Gln Asn Gln Pro Thr Gly Leu Met Ser Val Pro Asn Ala Leu 210 215 220 Thr Thr Gln Gln Gln Gln Gln Gln Lys Leu Arg Leu Gln Arg Ile Gln 225 230 235 240 Met Glu Arg Glu Arg Ile Arg Met Arg Gln Glu Glu Leu Met Arg Gln 245 250 255 Glu Ala Ala Leu Cys Arg Gln Leu Pro Met Glu Thr Glu Thr Met Ala 260 265 270 Pro Val Asn Thr Pro Ala Met Ser Thr Asp Met Arg Ser Val Thr Asn 275 280 285 Ser Ser Ser Asp Pro Phe Leu Asn Gly Gly Pro Tyr His Ser Arg Glu 290 295 300 Gln Ser Thr Asp Ser Gly Leu Gly Leu Gly Cys Tyr Ser Val Pro Thr 305 310 315 320 Thr Pro Glu Asp Phe Leu Ser Asn Met Asp Glu Met Asp Thr Gly Glu 325 330 335 Asn Ser Gly Gln Thr Pro Met Thr Val Asn Pro Gln Gln Thr Arg Phe 340 345 350 Pro Asp Phe Leu Asp Cys Leu Pro Gly Thr Asn Val Asp Leu Gly Thr 355 360 365 Leu Glu Ser Glu Asp Leu Ile Pro Leu Phe Asn Asp Val Glu Ser Ala 370 375 380 Leu Asn Lys Ser Glu Pro Phe Leu Thr Trp Leu 385 390 395 <210> 9 <211> 521 <212> PRT <213> RUNX-HUMAN <400> 9 Met Ala Ser Asn Ser Leu Phe Ser Thr Val Thr Pro Cys Gln Gln Asn 1 5 10 15 Phe Phe Trp Asp Pro Ser Thr Ser Arg Arg Phe Ser Pro Pro Ser Ser 20 25 30 Ser Leu Gln Pro Gly Lys Met Ser Asp Val Ser Pro Val Val Ala Ala 35 40 45 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 50 55 60 Gln Gln Gln Gln Gln Gln Gln Glu Ala Ala Ala Ala Ala Ala Ala Ala 65 70 75 80 Ala Ala Ala Ala Ala Ala Ala Ala Ala Val Pro Arg Leu Arg Pro Pro 85 90 95 His Asp Asn Arg Thr Met Val Glu Ile Ile Ala Asp His Pro Ala Glu 100 105 110 Leu Val Arg Thr Asp Ser Pro Asn Phe Leu Cys Ser Val Leu Pro Ser 115 120 125 His Trp Arg Cys Asn Lys Thr Leu Pro Val Ala Phe Lys Val Val Ala 130 135 140 Leu Gly Glu Val Pro Asp Gly Thr Val Val Thr Val Met Ala Gly Asn 145 150 155 160 Asp Glu Asn Tyr Ser Ala Glu Leu Arg Asn Ala Ser Ala Val Met Lys 165 170 175 Asn Gln Val Ala Arg Phe Asn Asp Leu Arg Phe Val Gly Arg Ser Gly 180 185 190 Arg Gly Lys Ser Phe Thr Leu Thr Ile Thr Val Phe Thr Asn Pro Pro 195 200 205 Gln Val Ala Thr Tyr His Arg Ala Ile Lys Val Thr Val Asp Gly Pro 210 215 220 Arg Glu Pro Arg Arg His Arg Gln Lys Leu Asp Asp Ser Lys Pro Ser 225 230 235 240 Leu Phe Ser Asp Arg Leu Ser Asp Leu Gly Arg Ile Pro His Pro Ser 245 250 255 Met Arg Val Gly Val Pro Pro Gln Asn Pro Arg Pro Ser Leu Asn Ser 260 265 270 Ala Pro Ser Pro Phe Asn Pro Gln Gly Gln Ser Gln Ile Thr Asp Pro 275 280 285 Arg Gln Ala Gln Ser Ser Pro Pro Trp Ser Tyr Asp Gln Ser Tyr Pro 290 295 300 Ser Tyr Leu Ser Gln Met Thr Ser Pro Ser Ile His Ser Thr Thr Pro 305 310 315 320 Leu Ser Ser Thr Arg Gly Thr Gly Leu Pro Ala Ile Thr Asp Val Pro 325 330 335 Arg Arg Ile Ser Asp Asp Asp Thr Ala Thr Ser Asp Phe Cys Leu Trp 340 345 350 Pro Ser Thr Leu Ser Lys Lys Ser Gln Ala Gly Ala Ser Glu Leu Gly 355 360 365 Pro Phe Ser Asp Pro Arg Gln Phe Pro Ser Ile Ser Ser Leu Thr Glu 370 375 380 Ser Arg Phe Ser Asn Pro Arg Met His Tyr Pro Ala Thr Phe Thr Tyr 385 390 395 400 Thr Pro Pro Val Thr Ser Gly Met Ser Leu Gly Met Ser Ala Thr Thr 405 410 415 His Tyr His Thr Tyr Leu Pro Pro Pro Tyr Pro Gly Ser Ser Gln Ser 420 425 430 Gln Ser Gly Pro Phe Gln Thr Ser Ser Thr Pro Tyr Leu Tyr Tyr Gly 435 440 445 Thr Ser Ser Gly Ser Tyr Gln Phe Pro Met Val Pro Gly Gly Asp Arg 450 455 460 Ser Pro Ser Arg Met Leu Pro Pro Cys Thr Thr Thr Ser Asn Gly Ser 465 470 475 480 Thr Leu Leu Asn Pro Asn Leu Pro Asn Gln Asn Asp Gly Val Asp Ala 485 490 495 Asp Gly Ser His Ser Ser Ser Pro Thr Val Leu Asn Ser Ser Gly Arg 500 505 510 Met Asp Glu Ser Val Trp Arg Pro Tyr 515 520 <210> 10 <211> 607 <212> PRT <213> RUNX-MOUSE <400> 10 Met Leu His Ser Pro His Lys Gln Pro Gln Asn His Lys Cys Gly Ala 1 5 10 15 Asn Phe Leu Gln Glu Asp Cys Lys Lys Ala Leu Ala Phe Lys Trp Leu 20 25 30 Ile Ser Ala Gly His Tyr Gln Pro Pro Arg Pro Thr Glu Ser Val Ser 35 40 45 Ala Leu Thr Thr Val His Ala Gly Ile Phe Lys Ala Ala Ser Ser Ile 50 55 60 Tyr Asn Arg Gly His Lys Phe Tyr Leu Glu Lys Lys Gly Gly Thr Met 65 70 75 80 Ala Ser Asn Ser Leu Phe Ser Ala Val Thr Pro Cys Gln Gln Ser Phe 85 90 95 Phe Trp Asp Pro Ser Thr Ser Arg Arg Phe Ser Pro Pro Ser Ser Ser 100 105 110 Leu Gln Pro Gly Lys Met Ser Asp Val Ser Pro Val Val Ala Ala Gln 115 120 125 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 130 135 140 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu Ala Ala Ala 145 150 155 160 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Val 165 170 175 Pro Arg Leu Arg Pro Pro His Asp Asn Arg Thr Met Val Glu Ile Ile 180 185 190 Ala Asp His Pro Ala Glu Leu Val Arg Thr Asp Ser Pro Asn Phe Leu 195 200 205 Cys Ser Val Leu Pro Ser His Trp Arg Cys Asn Lys Thr Leu Pro Val 210 215 220 Ala Phe Lys Val Val Ala Leu Gly Glu Val Pro Asp Gly Thr Val Val 225 230 235 240 Thr Val Met Ala Gly Asn Asp Glu Asn Tyr Ser Ala Glu Leu Arg Asn 245 250 255 Ala Ser Ala Val Met Lys Asn Gln Val Ala Arg Phe Asn Asp Leu Arg 260 265 270 Phe Val Gly Arg Ser Gly Arg Gly Lys Ser Phe Thr Leu Thr Ile Thr 275 280 285 Val Phe Thr Asn Pro Pro Gln Val Ala Thr Tyr His Arg Ala Ile Lys 290 295 300 Val Thr Val Asp Gly Pro Arg Glu Pro Arg Arg His Arg Gln Lys Leu 305 310 315 320 Asp Asp Ser Lys Pro Ser Leu Phe Ser Asp Arg Leu Ser Asp Leu Gly 325 330 335 Arg Ile Pro His Pro Ser Met Arg Val Gly Val Pro Pro Gln Asn Pro 340 345 350 Arg Pro Ser Leu Asn Ser Ala Pro Ser Pro Phe Asn Pro Gln Gly Gln 355 360 365 Ser Gln Ile Thr Asp Pro Arg Gln Ala Gln Ser Ser Pro Pro Trp Ser 370 375 380 Tyr Asp Gln Ser Tyr Pro Ser Tyr Leu Ser Gln Met Thr Ser Pro Ser 385 390 395 400 Ile His Ser Thr Thr Pro Leu Ser Ser Thr Arg Gly Thr Gly Leu Pro 405 410 415 Ala Ile Thr Asp Val Pro Arg Arg Ile Ser Asp Asp Asp Thr Ala Thr 420 425 430 Ser Asp Phe Cys Leu Trp Pro Ser Ser Leu Ser Lys Lys Ser Gln Ala 435 440 445 Gly Ala Ser Glu Leu Gly Pro Phe Ser Asp Pro Arg Gln Phe Pro Ser 450 455 460 Ile Ser Ser Leu Thr Glu Ser Arg Phe Ser Asn Pro Arg Met His Tyr 465 470 475 480 Pro Ala Thr Phe Thr Tyr Thr Pro Pro Val Thr Ser Gly Met Ser Leu 485 490 495 Gly Met Ser Ala Thr Thr His Tyr His Thr Tyr Leu Pro Pro Pro Tyr 500 505 510 Pro Gly Ser Ser Gln Ser Gln Ser Gly Pro Phe Gln Thr Ser Ser Thr 515 520 525 Pro Tyr Leu Tyr Tyr Gly Thr Ser Ser Ala Ser Tyr Gln Phe Pro Met 530 535 540 Val Pro Gly Gly Asp Arg Ser Pro Ser Arg Met Val Pro Pro Cys Thr 545 550 555 560 Thr Thr Ser Asn Gly Ser Thr Leu Leu Asn Pro Asn Leu Pro Asn Gln 565 570 575 Asn Asp Gly Val Asp Ala Asp Gly Ser His Ser Ser Ser Pro Thr Val 580 585 590 Leu Asn Ser Ser Gly Arg Met Asp Glu Ser Val Trp Arg Pro Tyr 595 600 605 <210> 11 <211> 457 <212> PRT <213> LMP-1 HUMAN <400> 11 Met Asp Ser Phe Lys Val Val Leu Glu Gly Pro Ala Pro Trp Gly Phe 1 5 10 15 Arg Leu Gln Gly Gly Lys Asp Phe Asn Val Pro Leu Ser Ile Ser Arg 20 25 30 Leu Thr Pro Gly Gly Lys Ala Ala Gln Ala Gly Val Ala Val Gly Asp 35 40 45 Trp Val Leu Ser Ile Asp Gly Glu Asn Ala Gly Ser Leu Thr His Ile 50 55 60 Glu Ala Gln Asn Lys Ile Arg Ala Cys Gly Glu Arg Leu Ser Leu Gly 65 70 75 80 Leu Ser Arg Ala Gln Pro Val Gln Ser Lys Pro Gln Lys Ala Ser Ala 85 90 95 Pro Ala Ala Asp Pro Pro Arg Tyr Thr Phe Ala Pro Ser Val Ser Leu 100 105 110 Asn Lys Thr Ala Arg Pro Phe Gly Ala Pro Pro Pro Ala Asp Ser Ala 115 120 125 Pro Gln Gln Asn Gly Gln Pro Leu Arg Pro Leu Val Pro Asp Ala Ser 130 135 140 Lys Gln Arg Leu Met Glu Asn Thr Glu Asp Trp Arg Pro Arg Pro Gly 145 150 155 160 Thr Gly Gln Ser Arg Ser Phe Arg Ile Leu Ala His Leu Thr Gly Thr 165 170 175 Glu Phe Met Gln Asp Pro Asp Glu Glu His Leu Lys Lys Ser Ser Gln 180 185 190 Val Pro Arg Thr Glu Ala Pro Ala Pro Ala Ser Ser Thr Pro Gln Glu 195 200 205 Pro Trp Pro Gly Pro Thr Ala Pro Ser Pro Thr Ser Arg Pro Pro Trp 210 215 220 Ala Val Asp Pro Ala Phe Ala Glu Arg Tyr Ala Pro Asp Lys Thr Ser 225 230 235 240 Thr Val Leu Thr Arg His Ser Gln Pro Ala Thr Pro Thr Pro Leu Gln 245 250 255 Ser Arg Thr Ser Ile Val Gln Ala Ala Ala Gly Gly Val Pro Gly Gly 260 265 270 Gly Ser Asn Asn Gly Lys Thr Pro Val Cys His Gln Cys His Lys Val 275 280 285 Ile Arg Gly Arg Tyr Leu Val Ala Leu Gly His Ala Tyr His Pro Glu 290 295 300 Glu Phe Val Cys Ser Gln Cys Gly Lys Val Leu Glu Glu Gly Gly Phe 305 310 315 320 Phe Glu Glu Lys Gly Ala Ile Phe Cys Pro Pro Cys Tyr Asp Val Arg 325 330 335 Tyr Ala Pro Ser Cys Ala Lys Cys Lys Lys Lys Ile Thr Gly Glu Ile 340 345 350 Met His Ala Leu Lys Met Thr Trp His Val His Cys Phe Thr Cys Ala 355 360 365 Ala Cys Lys Thr Pro Ile Arg Asn Arg Ala Phe Tyr Met Glu Glu Gly 370 375 380 Val Pro Tyr Cys Glu Arg Asp Tyr Glu Lys Met Phe Gly Thr Lys Cys 385 390 395 400 His Gly Cys Asp Phe Lys Ile Asp Ala Gly Asp Arg Phe Leu Glu Ala 405 410 415 Leu Gly Phe Ser Trp His Asp Thr Cys Phe Val Cys Ala Ile Cys Gln 420 425 430 Ile Asn Leu Glu Gly Lys Thr Phe Tyr Ser Lys Lys Asp Arg Pro Leu 435 440 445 Cys Lys Ser His Ala Phe Ser His Val 450 455 <210> 12 <211> 457 <212> PRT <213> LMP-1 MOUSE <400> 12 Met Asp Ser Phe Lys Val Val Leu Glu Gly Pro Ala Pro Trp Gly Phe 1 5 10 15 Arg Leu Gln Gly Gly Lys Asp Phe Asn Val Pro Leu Ser Ile Ser Arg 20 25 30 Leu Thr Pro Gly Gly Lys Ala Ala Gln Ala Gly Val Ala Val Gly Asp 35 40 45 Trp Val Leu Asn Ile Asp Gly Glu Asn Ala Gly Ser Leu Thr His Ile 50 55 60 Glu Ala Gln Asn Lys Ile Arg Ala Cys Gly Glu Arg Leu Ser Leu Gly 65 70 75 80 Leu Ser Arg Ala Gln Pro Val Gln Ser Lys Pro Gln Lys Ala Leu Thr 85 90 95 Pro Pro Ala Asp Pro Pro Arg Tyr Thr Phe Ala Pro Ser Ala Ser Leu 100 105 110 Asn Lys Thr Ala Arg Pro Phe Gly Ala Pro Pro Pro Thr Asp Ser Thr 115 120 125 Leu Arg Gln Asn Gly Gln Leu Leu Arg Gln Pro Val Pro Asp Ala Ser 130 135 140 Lys Gln Arg Leu Met Glu Asp Thr Glu Asp Trp Arg Pro Arg Pro Gly 145 150 155 160 Thr Gly Gln Ser Arg Ser Phe Arg Ile Leu Ala His Leu Thr Gly Thr 165 170 175 Glu Phe Met Gln Asp Pro Asp Glu Glu Phe Met Lys Lys Ser Ser Gln 180 185 190 Val Pro Arg Thr Glu Ala Pro Ala Pro Ala Ser Thr Ile Pro Gln Glu 195 200 205 Ser Trp Pro Gly Pro Thr Thr Pro Ser Pro Thr Ser Arg Pro Pro Trp 210 215 220 Ala Val Asp Pro Ala Phe Ala Glu Arg Tyr Ala Pro Asp Lys Thr Ser 225 230 235 240 Thr Val Leu Thr Arg His Ser Gln Pro Ala Thr Pro Thr Pro Leu Gln 245 250 255 Asn Arg Thr Ser Ile Val Gln Ala Ala Ala Gly Gly Gly Thr Gly Gly 260 265 270 Gly Ser Asn Asn Gly Lys Thr Pro Val Cys His Gln Cys His Lys Ile 275 280 285 Ile Arg Gly Arg Tyr Leu Val Ala Leu Gly His Ala Tyr His Pro Glu 290 295 300 Glu Phe Val Cys Ser Gln Cys Gly Lys Val Leu Glu Glu Gly Gly Phe 305 310 315 320 Phe Glu Glu Lys Gly Ala Ile Phe Cys Pro Ser Cys Tyr Asp Val Arg 325 330 335 Tyr Ala Pro Asn Cys Ala Lys Cys Lys Lys Lys Ile Thr Gly Glu Ile 340 345 350 Met His Ala Leu Lys Met Thr Trp His Val His Cys Phe Thr Cys Ala 355 360 365 Ala Cys Lys Thr Pro Ile Arg Asn Arg Ala Phe Tyr Met Glu Glu Gly 370 375 380 Ala Pro Tyr Cys Glu Arg Asp Tyr Glu Lys Met Phe Gly Thr Lys Cys 385 390 395 400 Arg Gly Cys Asp Phe Lys Ile Asp Ala Gly Asp Arg Phe Leu Glu Ala 405 410 415 Leu Gly Phe Ser Trp His Asp Thr Cys Phe Val Cys Ala Ile Cys Gln 420 425 430 Ile Asn Leu Glu Gly Lys Thr Phe Tyr Ser Lys Lys Asp Lys Pro Leu 435 440 445 Cys Lys Ser His Ala Phe Ser His Val 450 455 <110> Seoul National University Industry Foundation <120> Cell Permeable Fusion Protein for Strengthening Regenerative          Potential of Stem Cells &Lt; 130 > P11-B104 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 153 <212> PRT <213> SOD-HUMAN <400> 1 Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly   1 5 10 15 Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp              20 25 30 Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His          35 40 45 Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe      50 55 60 Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His  65 70 75 80 Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp                  85 90 95 Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile             100 105 110 Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys         115 120 125 Gly Gly Asn Glu Gly Ser Thr Lys Thr Gly Asn Gly Ser Arg Leu     130 135 140 Ala Cys Gly Val Ile Gly Ile Ala Gln 145 150 <210> 2 <211> 103 <212> PRT <213> Thioredoxin-HUMAN <400> 2 Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala   1 5 10 15 Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly              20 25 30 Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys Tyr          35 40 45 Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp Val      50 55 60 Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe Lys  65 70 75 80 Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu                  85 90 95 Glu Ala Thr Ile Asn Glu Leu             100 <210> 3 <211> 277 <212> PRT <213> HOX4 <400> 3 Met Lys Arg Pro Gly Gly Ala Gly Gly Gly Gly Gly Ser Pro Ser Leu   1 5 10 15 Val Thr Met Ala Asn Ser Ser Asp Asp Gly Tyr Gly Gly Val Gly Met              20 25 30 Glu Ala Glu Gly Asp Val Glu Glu Glu Met Met Ala Cys Gly Gly Gly          35 40 45 Gly Glu Lys Lys Arg Arg Leu Ser Val Glu Gln Val Arg Ala Leu Glu      50 55 60 Arg Ser Phe Glu Val Glu Asn Lys Leu Glu Pro Glu Arg Lys Ala Arg  65 70 75 80 Leu Ala Arg Asp Leu Gly Leu Gln Pro Arg Gln Val Ala Val Trp Phe                  85 90 95 Gln Asn Arg Arg Ala Arg Trp Lys Thr Lys Gln Leu Glu Arg Asp Tyr             100 105 110 Ala Ala Leu Arg His Ser Tyr Asp Ser Leu Arg Leu Asp His Asp Ala         115 120 125 Leu Arg Arg Asp Lys Asp Ala Leu Leu Ala Glu Ile Lys Glu Leu Lys     130 135 140 Ala Lys Leu Gly Asp Glu Glu Ala Ala Ala Ser Phe Thr Ser Val Lys 145 150 155 160 Glu Glu Pro Ala Ala Ser Asp Gly Pro Pro Ala Ala Gly Phe Gly Ser                 165 170 175 Ser Asp Ser Ser Ser Ala Val Leu Asn Asp Val Asp Ala Ala Gly             180 185 190 Ala Ala Pro Ala Ala Thr Asp Ala Leu Ala Pro Glu Ala Cys Thr Phe         195 200 205 Leu Gly Ala Pro Ala Ala Gla Ala Gla Ala Gla Ala Ala Ala     210 215 220 Ala Ser His Glu Glu Val Phe Phe His Gly Asn Phe Leu Lys Val Glu 225 230 235 240 Glu Asp Glu Thr Gly Phe Leu Asp Asp Asp Glu Pro Cys Gly Gly Phe                 245 250 255 Phe Ala Asp Asp Gln Pro Pro Pro Leu Ser Ser Trp Trp Ala Glu Pro             260 265 270 Thr Glu His Trp Asn         275 <210> 4 <211> 347 <212> PRT <213> NF-Ya <400> 4 Met Glu Gln Tyr Thr Ala Asn Ser Asn Ser Ser Thr Glu Gln Ile Val   1 5 10 15 Val Gln Ala Gly Gln Ile Gln Gln Gln Gln Gln Gly Gly Val Thr Ala              20 25 30 Val Gln Leu Gln Thr Glu Ala Gln Val Ala Ser Ala Ser Gly Gln Gln          35 40 45 Val Gln Thr Leu Gln Val Val Gln Gly Gln Pro Leu Met Val Gln Val      50 55 60 Ser Gly Gly Gln Leu Ile Thr Ser Thr Gly Gln Pro Ile Met Val Gln  65 70 75 80 Ala Val Pro Gly Gly Gln Gly Gln Thr Ile Met Gln Val Pro Val Ser                  85 90 95 Gly Thr Gln Gly Leu Gln Gln Ile Gln Leu Val Pro Pro Gly Gln Ile             100 105 110 Gln Ile Gln Gln Gln Gln Ala Val Gln Val Gln Gln Gln Gln Gly Gln         115 120 125 Thr Gln Gln Ile Ile Gln Gln Pro Gln Thr Ala Val Thr Ala Gly     130 135 140 Gln Thr Gln Thr Gln Gln Gln Ile Ala Val Gln Gly Gln Gln Val Ala 145 150 155 160 Gln Thr Ala Glu Gly Gln Thr Ile Val Tyr Gln Pro Val Asn Ala Asp                 165 170 175 Gly Thr Ile Leu Gln Gln Val Thr Val Val Ser Gly Met Ile Thr             180 185 190 Ile Pro Ala Ala Ser Leu Ala Gly Ala Gln Ile Val Gln Thr Gly Ala         195 200 205 Asn Thr Asn Thr Ser Ser Gly Gln Gly Thr Val Thr Val Thr Leu     210 215 220 Pro Val Ala Gly As Val Val Asn Ser Gly Gly Met Val Met Met Val 225 230 235 240 Pro Gly Ala Gly Ser Val Pro Ala Ile Gln Arg Ile Pro Leu Pro Gly                 245 250 255 Ala Glu Met Leu Glu Glu Glu Pro Leu Tyr Val Asn Ala Lys Gln Tyr             260 265 270 His Arg Ile Leu Lys Arg Arg Gln Ala Arg Ala Lys Leu Glu Ala Glu         275 280 285 Gly Lys Ile Pro Lys Glu Arg Arg Lys Tyr Leu His Glu Ser Arg His     290 295 300 Arg His Ala Met Ala Arg Lys Arg Gly Glu Gly Gly Arg Phe Phe Ser 305 310 315 320 Pro Lys Glu Lys Asp Ser Pro His Met Gln Asp Pro Asn Gln Ala Asp                 325 330 335 Glu Glu Ala Met Thr Gln Ile Ile Arg Val Ser             340 345 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> TAT (Transactivator of Transcription Peptide from HIV type-1) <400> 5 Cys Gly Arg Lys Lys Arg Arg Gln Arg Arg Pro Pro Gln Cys   1 5 10 15 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Penetratin <400> 6 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 <210> 7 <211> 400 <212> PRT <213> TAZ-HUMAN <400> 7 Met Asn Pro Ala Ser Ala Pro Pro Leu Pro Pro Pro Gly Gln Gln   1 5 10 15 Val Ile His Val Thr Gln Asp Leu Asp Thr Asp Leu Glu Ala Leu Phe              20 25 30 Asn Ser Val Met Asn Pro Lys Pro Ser Ser Trp Arg Lys Lys Ile Leu          35 40 45 Pro Glu Ser Phe Phe Lys Glu Pro Asp Ser Ser Ser Ser Ser Gln      50 55 60 Ser Ser Thr Asp Ser Ser Gly Gly His Pro Gly Pro Arg Leu Ala Gly  65 70 75 80 Gly Ala Gln His Val Arg Ser His Ser Ser Pro Ala Ser Leu Gln Leu                  85 90 95 Gly Thr Gly Ala Gly Ala Gly Ser Pro Ala Gln Gln His Ala His             100 105 110 Leu Arg Gln Gln Ser Tyr Asp Val Thr Asp Glu Leu Pro Leu Pro Pro         115 120 125 Gly Trp Glu Met Thr Phe Thr Ala Thr Gly Gln Arg Tyr Phe Leu Asn     130 135 140 His Ile Glu Lys Ile Thr Thr Trp Gln Asp Pro Arg Lys Ala Met Asn 145 150 155 160 Gln Pro Leu Asn His Met Asn Leu His Pro Ala Val Ser Ser Thr Pro                 165 170 175 Val Pro Gln Arg Ser Met Ala Val Ser Gln Pro Asn Leu Val Met Asn             180 185 190 His Gln His Gln Gln Gln Met Ala Pro Ser Thr Leu Ser Gln Gln Asn         195 200 205 His Pro Thr Gln Asn Pro Pro Ala Gly Leu Met Ser Met Pro Asn Ala     210 215 220 Leu Thr Thr Gln Gln Gln Gln Gln Gln Lys Leu Arg Leu Gln Arg Ile 225 230 235 240 Gln Met Glu Arg Glu Arg Ile Arg Met Met Arg Gln Glu Glu Leu Met Arg                 245 250 255 Gln Glu Ala Ala Leu Cys Arg Gln Leu Pro Met Glu Ala Glu Thr Leu             260 265 270 Ala Pro Val Gln Ala Ala Val Asn Pro Pro Thr Met Thr Pro Asp Met         275 280 285 Arg Ser Ile Thr Asn Ser Ser Asp Pro Phe Leu Asn Gly Gly Pro     290 295 300 Tyr His Ser Arg Glu Gln Ser Thr Asp Ser Gly Leu Gly Leu Gly Cys 305 310 315 320 Tyr Ser Val Pro Thr Thr Pro Glu Asp Phe Leu Ser Asn Val Asp Glu                 325 330 335 Met Asp Thr Gly Glu Asn Ala Gly Gln Thr Pro Met Asn Ile Asn Pro             340 345 350 Gln Gln Thr Arg Phe Pro Asp Phe Leu Asp Cys Leu Pro Gly Thr Asn         355 360 365 Val Asp Leu Gly Thr Leu Glu Ser Glu Asp Leu Ile Pro Leu Phe Asn     370 375 380 Asp Val Glu Ser Ala Leu Asn Lys Ser Glu Pro Phe Leu Thr Trp Leu 385 390 395 400 <210> 8 <211> 395 <212> PRT <213> TAZ-MOUSE <400> 8 Met Asn Pro Ser Ser Val Pro His Pro Leu Pro Pro Pro Gly Gln Gln   1 5 10 15 Val Ile His Val Thr Gln Asp Leu Asp Thr Asp Leu Glu Ala Leu Phe              20 25 30 Asn Ser Val Met Asn Pro Lys Pro Ser Ser Trp Arg Lys Lys Ile Leu          35 40 45 Pro Glu Ser Phe Phe Lys Glu Pro Asp Ser Ser Ser Ser Ser Gln      50 55 60 Ser Ser Thr Asp Ser Ser Gly Gly His Pro Gly Pro Arg Leu Ala Gly  65 70 75 80 Gly Ala Gln His Val Arg Ser His Ser Ser Pro Ala Ser Leu Gln Leu                  85 90 95 Gly Thr Gly Ala Gly Ala Ala Gly Gly Pro Ala Gln Gln His Ala His             100 105 110 Leu Arg Gln Gln Ser Tyr Asp Val Thr Asp Glu Leu Pro Leu Pro Pro         115 120 125 Gly Trp Glu Met Thr Phe Thr Ala Thr Gly Gln Arg Tyr Phe Leu Asn     130 135 140 His Ile Glu Lys Ile Thr Thr Trp Gln Asp Pro Arg Lys Val Met Asn 145 150 155 160 Gln Pro Leu Asn His Val Asn Leu His Pro Ser Ile Thr Ser Thr Ser                 165 170 175 Val Pro Gln Arg Ser Met Ala Val Ser Gln Pro Asn Leu Ala Met Asn             180 185 190 His Gln His Gln Gln Val Val Ala Thr Ser Leu Ser Pro Gln Asn His         195 200 205 Pro Thr Gln Asn Gln Pro Thr Gly Leu Met Ser Val Pro Asn Ala Leu     210 215 220 Thr Gln Gln Gln Gln Gln Gln Lys Leu Arg Leu Gln Arg Ile Gln 225 230 235 240 Met Glu Arg Glu Arg Ile Arg Met Met Arg Gln Glu Glu Leu Met Arg Gln                 245 250 255 Glu Ala Ala Leu Cys Arg Gln Leu Pro Met Glu Thr Glu Thr Met Ala             260 265 270 Pro Val Asn Thr Pro Ala Met Ser Thr Asp Met Arg Ser Val Thr Asn         275 280 285 Ser Ser Asp Pro Phe Leu Asn Gly Gly Pro Tyr His Ser Arg Glu     290 295 300 Gln Ser Thr Asp Ser Gly Leu Gly Leu Gly Cys Tyr Ser Val Pro Thr 305 310 315 320 Thr Pro Glu Asp Phe Leu Ser Asn Met Asp Glu Met Asp Thr Gly Glu                 325 330 335 Asn Ser Gly Gln Thr Pro Met Thr Val Asn Pro Gln Gln Thr Arg Phe             340 345 350 Pro Asp Phe Leu Asp Cys Leu Pro Gly Thr Asn Val Asp Leu Gly Thr         355 360 365 Leu Glu Ser Glu Asp Leu Ile Pro Leu Phe Asn Asp Val Glu Ser Ala     370 375 380 Leu Asn Lys Ser Glu Pro Phe Leu Thr Trp Leu 385 390 395 <210> 9 <211> 521 <212> PRT <213> RUNX-HUMAN <400> 9 Met Ala Ser Asn Ser Leu Phe Ser Thr Val Thr Pro Cys Gln Gln Asn   1 5 10 15 Phe Phe Trp Asp Pro Ser Thr Ser Arg Arg Phe Ser Pro Pro Ser Ser              20 25 30 Ser Leu Gln Pro Gly Lys Met Ser Ser Val Val Ser Ala Val          35 40 45 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln      50 55 60 Gln Gln Gln Gln Gln Gln Gln Glu Ala Ala Ala Ala Ala Ala Ala Ala  65 70 75 80 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Val                  85 90 95 His Asp Asn Arg Thr Met Val Glu Ile Ile Ala Asp His Pro Ala Glu             100 105 110 Leu Val Arg Thr Asp Ser Pro Asn Phe Leu Cys Ser Val Leu Pro Ser         115 120 125 His Trp Arg Cys Asn Lys Thr Leu Pro Val Ala Phe Lys Val Val Ala     130 135 140 Leu Gly Glu Val Pro Asp Gly Thr Val Val Thr Val Met Ala Gly Asn 145 150 155 160 Asp Glu Asn Tyr Ser Ala Glu Leu Arg Asn Ala Ser Ala Val Met Lys                 165 170 175 Asn Gln Val Ala Arg Phe Asn Asp Leu Arg Phe Val Gly Arg Ser Gly             180 185 190 Arg Gly Lys Ser Phe Thr Leu Thr Ile Thr Val Phe Thr Asn Pro Pro         195 200 205 Gln Val Ala Thr Tyr His Arg Ala Ile Lys Val Thr Val Asp Gly Pro     210 215 220 Arg Glu Pro Arg Arg His Arg Gln Lys Leu Asp Asp Ser Lys Pro Ser 225 230 235 240 Leu Phe Ser Asp Arg Leu Ser Asp Leu Gly Arg Ile Pro His Ser Ser                 245 250 255 Met Arg Val Gly Val Pro Pro Gln Asn Pro Arg Pro Ser Leu Asn Ser             260 265 270 Ala Pro Ser Pro Phe Asn Pro Gln Gly Gln Ser Gln Ile Thr Asp Pro         275 280 285 Arg Gln Ala Gln Ser Ser Pro Pro Trp Ser Tyr Asp Gln Ser Tyr Pro     290 295 300 Ser Tyr Leu Ser Gln Met Thr Ser Pro Ser Ile His Ser Thr Thr Pro 305 310 315 320 Leu Ser Ser Thr Arg Gly Thr Gly Leu Pro Ala Ile Thr Asp Val Pro                 325 330 335 Arg Arg Ile Ser Asp Asp Asp Thr Ala Thr Ser Asp Phe Cys Leu Trp             340 345 350 Pro Ser Thr Leu Ser Lys Lys Ser Gln Ala Gly Ala Ser Glu Leu Gly         355 360 365 Pro Phe Ser Asp Pro Arg Gln Phe Pro Ser Ser Ser Seru Thr Glu     370 375 380 Ser Arg Phe Ser Asn Pro Arg Met His Tyr Pro Ala Thr Phe Thr Tyr 385 390 395 400 Thr Pro Pro Val Thr Ser Gly Met Ser Leu Gly Met Ser Ala Thr Thr                 405 410 415 His Tyr His Thr Tyr Leu Pro Pro Pro Tyr Pro Gly Ser Ser Gln Ser             420 425 430 Gln Ser Gly Pro Phe Gln Thr Ser Ser Thr Pro Tyr Leu Tyr Tyr Gly         435 440 445 Thr Ser Ser Gly Ser Tyr Gln Phe Pro Met Val Pro Gly Gly Asp Arg     450 455 460 Ser Pro Ser Arg Met Leu Pro Pro Cys Thr Thr Thr Ser Asn Gly Ser 465 470 475 480 Thr Leu Leu Asn Pro Asn Leu Pro Asn Gln Asn Asp Gly Val Asp Ala                 485 490 495 Asp Gly Ser His Ser Ser Ser Pro Thr Val Leu Asn Ser Ser Gly Arg             500 505 510 Met Asp Glu Ser Val Trp Arg Pro Tyr         515 520 <210> 10 <211> 607 <212> PRT <213> RUNX-MOUSE <400> 10 Met Leu His Ser Pro His Lys Gln Pro Gln Asn His Lys Cys Gly Ala   1 5 10 15 Asn Phe Leu Gln Glu Asp Cys Lys Lys Ala Leu Ala Phe Lys Trp Leu              20 25 30 Ile Ser Ala Gly His Tyr Gln Pro Pro Arg Pro Thr Glu Ser Val Ser          35 40 45 Ala Leu Thr Thr Val Ala Gly Ile Phe Lys Ala Ala Ser Ser Ile      50 55 60 Tyr Asn Arg Gly His Lys Phe Tyr Leu Glu Lys Lys Gly Gly Thr Met  65 70 75 80 Ala Ser Asn Ser Leu Phe Ser Ala Val Thr Pro Cys Gln Gln Ser Phe                  85 90 95 Phe Trp Asp Pro Ser Thr Ser Arg Arg Phe Ser Pro Pro Ser Ser Ser             100 105 110 Leu Gln Pro Gly Lys Met Ser Ser Val Val Ser Ala Gln         115 120 125 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln     130 135 140 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu Ala Ala Ala 145 150 155 160 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala                 165 170 175 Pro Arg Leu Arg Pro Pro His Asp Asn Arg Thr Met Val Glu Ile Ile             180 185 190 Ala Asp His Pro Ala Glu Leu Val Arg Thr Asp Ser Pro Asn Phe Leu         195 200 205 Cys Ser Val Leu Pro Ser His Trp Arg Cys Asn Lys Thr Leu Pro Val     210 215 220 Ala Phe Lys Val Val Ala Leu Gly Glu Val Pro Asp Gly Thr Val Val 225 230 235 240 Thr Val Met Ala Gly Asn Asp Glu Asn Tyr Ser Ala Glu Leu Arg Asn                 245 250 255 Ala Ser Ala Val Met Lys Asn Gln Val Ala Arg Phe Asn Asp Leu Arg             260 265 270 Phe Val Gly Arg Ser Gly Arg Gly Lys Ser Phe Thr Leu Thr Ile Thr         275 280 285 Val Phe Thr Asn Pro Pro Gln Val Ala Thr Tyr His Arg Ala Ile Lys     290 295 300 Val Thr Val Asp Gly Pro Arg Glu Pro Arg Arg His Arg Gln Lys Leu 305 310 315 320 Asp Asp Ser Lys Pro Ser Leu Phe Ser Asp Arg Leu Ser Asp Leu Gly                 325 330 335 Arg Ile Pro His Pro Ser Met Arg Val Gly Val Pro Pro Gln Asn Pro             340 345 350 Arg Pro Ser Leu Asn Ser Ala Pro Ser Pro Phe Asn Pro Gln Gly Gln         355 360 365 Ser Gln Ile Thr Asp Pro Arg Gln Ala Gln Ser Ser Pro Pro Trp Ser     370 375 380 Tyr Asp Gln Ser Tyr Pro Ser Tyr Leu Ser Gln Met Thr Ser Pro Ser 385 390 395 400 Ile His Ser Thr Thr Pro Leu Ser Ser Thr Arg Gly Thr Gly Leu Pro                 405 410 415 Ala Ile Thr Asp Val Arg Arg Ile Ser Asp Asp Asp Thr Ala Thr             420 425 430 Ser Asp Phe Cys Leu Trp Pro Ser Ser Leu Ser Lys Lys Ser Gln Ala         435 440 445 Gly Ala Ser Glu Leu Gly Pro Phe Ser Asp Pro Arg Gln Phe Pro Ser     450 455 460 Ile Ser Ser Leu Thr Glu Ser Arg Phe Ser Asn Pro Arg Met His Tyr 465 470 475 480 Pro Ala Thr Phe Thr Tyr Thr Pro Pro Thal Ser Gly Met Ser Leu                 485 490 495 Gly Met Ser Ala Thr His Tyr His Thr Tyr Leu Pro Pro Pro Tyr             500 505 510 Pro Gly Ser Ser Gln Ser Gln Ser Gly Pro Phe Gln Thr Ser Ser Thr         515 520 525 Pro Tyr Leu Tyr Tyr Gly Thr Ser Ser Ala Ser Tyr Gln Phe Pro Met     530 535 540 Val Pro Gly Asp Arg Ser Pro Ser Arg Met Val Pro Pro Cys Thr 545 550 555 560 Thr Thr Ser Asn Gly Ser Thr Leu Leu Asn Pro Asn Leu Pro Asn Gln                 565 570 575 Asn Asp Gly Val Asp Ala Asp Gly Ser His Ser Ser Ser Pro Thr Val             580 585 590 Leu Asn Ser Ser Gly Arg Met Asp Glu Ser Val Trp Arg Pro Tyr         595 600 605 <210> 11 <211> 457 <212> PRT <213> LMP-1 HUMAN <400> 11 Met Asp Ser Phe Lys Val Val Leu Glu Gly Pro Ala Pro Trp Gly Phe   1 5 10 15 Arg Leu Gln Gly Gly Lys Asp Phe Asn Val Pro Leu Ser Ile Ser Arg              20 25 30 Leu Thr Pro Gly Gly Lys Ala Ala Gln Ala Gly Val Ala Val Gly Asp          35 40 45 Trp Val Leu Ser Ile Asp Gly Glu Asn Ala Gly Ser Leu Thr His Ile      50 55 60 Glu Ala Gln Asn Lys Ile Arg Ala Cys Gly Glu Arg Leu Ser Leu Gly  65 70 75 80 Leu Ser Arg Ala Gln Pro Val Gln Ser Lys Pro Gln Lys Ala Ser Ala                  85 90 95 Pro Ala Ala Asp Pro Pro Arg Tyr Thr Phe Ala Pro Ser Val Ser Leu             100 105 110 Asn Lys Thr Ala Arg Pro Phe Gly Ala Pro Pro Ala Asp Ser Ala         115 120 125 Pro Gln Gln Asn Gly Gln Pro Leu Arg Pro Leu Val Pro Asp Ala Ser     130 135 140 Lys Gln Arg Leu Met Glu Asn Thr Glu Asp Trp Arg Pro Arg Pro Gly 145 150 155 160 Thr Gly Gln Ser Arg Ser Ser Phe Arg Ile Leu Ala His Leu Thr Gly Thr                 165 170 175 Glu Phe Met Gln Asp Pro Asp Glu Glu His Leu Lys Lys Ser Ser Gln             180 185 190 Val Pro Arg Thr Glu Ala Pro Ala Pro Ala Ser Ser Thr Pro Gln Glu         195 200 205 Pro Trp Pro Gly Pro Thr Ala Pro Ser Pro Thr Ser Arg Pro Pro Trp     210 215 220 Ala Val Asp Pro Ala Phe Ala Glu Arg Tyr Ala Pro Asp Lys Thr Ser 225 230 235 240 Thr Val Leu Thr Arg His Ser Gln Pro Ala Thr Pro Thr Pro Leu Gln                 245 250 255 Ser Arg Thr Ser Ile Val Gln Ala Ala Ala Gly Gly Val Pro Gly Gly             260 265 270 Gly Ser Asn Asn Gly Lys Thr Pro Val Cys His Gln Cys His Lys Val         275 280 285 Ile Arg Gly Arg Tyr Leu Val Ala Leu Gly His Ala Tyr His Pro Glu     290 295 300 Glu Phe Val Cys Ser Gln Cys Gly Lys Val Leu Glu Glu Gly Gly Phe 305 310 315 320 Phe Glu Glu Lys Gly Ala Ile Phe Cys Pro Pro Cys Tyr Asp Val Arg                 325 330 335 Tyr Ala Pro Ser Cys Ala Lys Cys Lys Lys Lys Ile Thr Gly Glu Ile             340 345 350 Met His Ala Leu Lys Met Thr Trp His Val His Cys Phe Thr Cys Ala         355 360 365 Ala Cys Lys Thr Pro Ile Arg Asn Arg Ala Phe Tyr Met Glu Glu Gly     370 375 380 Val Pro Tyr Cys Glu Arg Asp Tyr Glu Lys Met Phe Gly Thr Lys Cys 385 390 395 400 His Gly Cys Asp Phe Lys Ile Asp Ala Gly Asp Arg Phe Leu Glu Ala                 405 410 415 Leu Gly Phe Ser Trp His Asp Thr Cys Phe Val Cys Ala Ile Cys Gln             420 425 430 Ile Asn Leu Glu Gly Lys Thr Phe Tyr Ser Lys Lys Asp Arg Pro Leu         435 440 445 Cys Lys Ser His Ala Phe Ser His Val     450 455 <210> 12 <211> 457 <212> PRT <213> LMP-1 MOUSE <400> 12 Met Asp Ser Phe Lys Val Val Leu Glu Gly Pro Ala Pro Trp Gly Phe   1 5 10 15 Arg Leu Gln Gly Gly Lys Asp Phe Asn Val Pro Leu Ser Ile Ser Arg              20 25 30 Leu Thr Pro Gly Gly Lys Ala Ala Gln Ala Gly Val Ala Val Gly Asp          35 40 45 Trp Val Leu Asn Ile Asp Gly Glu Asn Ala Gly Ser Leu Thr His Ile      50 55 60 Glu Ala Gln Asn Lys Ile Arg Ala Cys Gly Glu Arg Leu Ser Leu Gly  65 70 75 80 Leu Ser Arg Ala Gln Pro Val Gln Ser Lys Pro Gln Lys Ala Leu Thr                  85 90 95 Pro Ala Asp Pro Pro Arg Tyr Thr Phe Ala Pro Ser Ala Ser Leu             100 105 110 Asn Lys Thr Ala Arg Pro Phe Gly Ala Pro Pro Thr Asp Ser Thr         115 120 125 Leu Arg Gln Asn Gly Gln Leu Leu Arg Gln Pro Val Pro Asp Ala Ser     130 135 140 Lys Gln Arg Leu Met Glu Asp Thr Glu Asp Trp Arg Pro Arg Pro Gly 145 150 155 160 Thr Gly Gln Ser Arg Ser Ser Phe Arg Ile Leu Ala His Leu Thr Gly Thr                 165 170 175 Glu Phe Met Gln Asp Pro Asp Glu Glu Phe Met Lys Lys Ser Ser Gln             180 185 190 Val Pro Arg Thr Glu Ala Pro Ala Pro Ala Ser Thr Ile Pro Gln Glu         195 200 205 Ser Trp Pro Gly Pro Thr Thr Pro Ser Pro Thr Ser Arg Pro Pro Trp     210 215 220 Ala Val Asp Pro Ala Phe Ala Glu Arg Tyr Ala Pro Asp Lys Thr Ser 225 230 235 240 Thr Val Leu Thr Arg His Ser Gln Pro Ala Thr Pro Thr Pro Leu Gln                 245 250 255 Asn Arg Thr Ser Ile Val Gln Ala Ala Gly Gly Gly Thr Gly Gly             260 265 270 Gly Ser Asn Asn Gly Lys Thr Pro Val Cys His Gln Cys His Lys Ile         275 280 285 Ile Arg Gly Arg Tyr Leu Val Ala Leu Gly His Ala Tyr His Pro Glu     290 295 300 Glu Phe Val Cys Ser Gln Cys Gly Lys Val Leu Glu Glu Gly Gly Phe 305 310 315 320 Phe Glu Glu Lys Gly Ala Ile Phe Cys Pro Ser Cys Tyr Asp Val Arg                 325 330 335 Tyr Ala Pro Asn Cys Ala Lys Cys Lys Lys Lys Ile Thr Gly Glu Ile             340 345 350 Met His Ala Leu Lys Met Thr Trp His Val His Cys Phe Thr Cys Ala         355 360 365 Ala Cys Lys Thr Pro Ile Arg Asn Arg Ala Phe Tyr Met Glu Glu Gly     370 375 380 Ala Pro Tyr Cys Glu Arg Asp Tyr Glu Lys Met Phe Gly Thr Lys Cys 385 390 395 400 Arg Gly Cys Asp Phe Lys Ile Asp Ala Gly Asp Arg Phe Leu Glu Ala                 405 410 415 Leu Gly Phe Ser Trp His Asp Thr Cys Phe Val Cys Ala Ile Cys Gln             420 425 430 Ile Asn Leu Glu Gly Lys Thr Phe Tyr Ser Lys Lys Asp Lys Pro Leu         435 440 445 Cys Lys Ser His Ala Phe Ser His Val     450 455

Claims (18)

Thioredoxin 항산화 단백질의 아미노 말단에 LMWP (Low Molecular Weight Protamine) 세포 침투성 펩타이드가 결합되어 있는 줄기세포의 근육세포로의 분화촉진능 향상용 세포 침투성 융합 단백질.
Thioredoxin Cell permeable fusion protein for enhancing the differentiation promoting activity of stem cells into muscle cells with LMWP (Low Molecular Weight Protamine) cell permeability peptide bound to the amino terminal of antioxidant protein.
삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 제1항에 있어서, 상기 Thioredoxin는 서열번호 2로 표시되는 것을 특징으로 하는 줄기세포의 근육세포로의 분화촉진능 향상용 세포 침투성 융합 단백질.
The cell permeable fusion protein according to claim 1, wherein the Thioredoxin is represented by SEQ ID NO: 2.
삭제delete 삭제delete 삭제delete LMWP (Low Molecular Weight Protamine) 세포 침투성 펩타이드 형질 도입 부위를 coding 하는 cDNA; Thioredoxin 항산화 단백질 형질 도입 부위를 coding 하는 cDNA; 및 6개의 히스티딘에 대한 cDNA를 포함하고 있으며, 줄기세포의 근육세포로의 분화촉진능을 가지는 융합 단백질의 발현 벡터.
CDNA coding for LMWP (Low Molecular Weight Protamine) cell penetrating peptide transduction site; CDNA encoding the thioredoxin antioxidant protein transduction site; And six histidine cDNAs, and expresses a fusion protein having the ability to promote the differentiation of stem cells into muscle cells.
제 11 항의 발현 벡터로 형질전환된 재조합 미생물.
12. A recombinant microorganism transformed with the expression vector of claim 11.
제12항의 재조합 미생물을 배양하여 줄기세포의 근육세포로의 분화촉진능을 가지는 융합 단백질을 발현시키는 단계; 및 상기 발현된 융합 단백질을 회수하는 단계를 포함하는 줄기세포의 근육세포로의 분화촉진능을 가지는 융합 단백질의 제조방법.
Culturing the recombinant microorganism of claim 12 to express a fusion protein having the ability to promote differentiation of stem cells into muscle cells; And recovering the expressed fusion protein, wherein the fusion protein has the ability to promote the differentiation of stem cells into muscle cells.
제13항의 방법에 의해 제조된 융합 단백질을 체외에서 (in vitro)에서 줄기세포에 도입하는 것을 특징으로 하는 줄기세포의 근육세포로의 분화촉진 방법.
A method for promoting differentiation of stem cells into muscle cells, characterized by introducing the fusion protein produced by the method of claim 13 into a stem cell in vitro.
삭제delete 삭제delete 삭제delete 삭제delete
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